Materials and methods for nerve grafting

ABSTRACT

The subject invention pertains to compositions and methods for promoting repair of damaged nerve tissue using nerve grafts and preparation of nerve grafts. The compositions and methods of the subject invention can be employed to restore the continuity of nerve interrupted by disease, traumatic events or surgical procedures. Compositions of the subject invention comprise one or more chondroitin sulfate proteoglycan (CSPG)-degrading enzymes that promote axonal penetration into damaged nerve tissue and nerve graft. The invention also concerns methods for promoting repair of damaged nerve tissue using the present compositions and nerve tissue treated according to such methods. The invention also includes storage solutions for nerve tissue.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of co-pending application Ser. No.13/776,606, filed Feb. 25, 2013; which is a continuation of Ser. No.12/190,359, filed Aug. 12, 2008; which is a continuation of Ser. No.10/218,315, filed Aug. 13, 2002; which claims the benefit of ProvisionalPatent Application Ser. No. 60/311,870, filed Aug. 13, 2001, thedisclosure of each which is hereby incorporated by reference herein intheir entirety, including all figures, tables, and drawings.

The subject invention was made with government support under a researchproject supported by National Institutes of Health Grant No. R01NS37901. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Peripheral nerve injuries are a major source of chronic disability. Poormanagement of nerve injuries is associated with muscle atrophy and canlead to painful neuroma when severed axons are unable to reestablishcontinuity with the distal nerve. Although nerves have the potential toregenerate after injury, this ability is strictly dependent upon theregenerating nerve fibers (and their axonal sprouts) making appropriatecontact with the severed nerve segment (and the Schwann cell basallaminae therein). Regenerating axons that fail to traverse the gap orinjury site and enter the basal lamina of the severed distal nervesegment will deteriorate, resulting in neuronal death, muscle atrophyand permanent functional deficit (Fawcett J W et al. [1990] Annu RevNeurosci 13:43-60).

Briefly, a nerve carries the peripheral processes (or axons) of neurons.The neuronal cell bodies reside in the spinal cord (motor neurons), inganglia situated along the vertebral column (spinal sensory ganglia) orin ganglia found throughout the organs of the body (autonomic andenteric ganglia). A nerve consists of axons, Schwann cells and extensiveconnective tissue sheaths (Dagum A B [1998] J Hand Ther 11:111-117). Theouter covering, the epineurium, is made of collagenous connective tissuethat cushions the fascicles from external pressure and surrounds theperineurium. The perineurium surrounds the individual fascicles and,together with endothelial cells in the endoneurial microvessels,functions as the blood-nerve barrier. The endoneurium lies inside theperineurium and consists of collagenous tissue that surrounds theSchwann cells and axons. A fascicular group consists of two or morefascicles surrounded, respectively, by perineurium and epineurium. Thetopography of nerves is constant distally, with a group of fasciclesbeing either sensory or motor. The neuron consists of a soma (cell body)and an axon, which can be several feet long.

In nerve injuries where there is axonal disruption, but the continuityof the endoneurial sheath remains intact (e.g., crush injury), axonsregenerate within their original basal lamina and complete recovery canbe expected. In contrast, axonal regrowth may be severely compromisedafter nerve transection and surgical repair is highly dependent on therealignment of the nerve elements described above (Dagum A B [1998] JHand Ther 11:111-117). Epineurial coaptation (neurorrhaphy) is theprimary method of dealing with nerve transection. However, the extent ofregeneration is highly variable and, at best, partial recovery offunction can be expected (Terzis J K et al. [1990] The Peripheral Nerve:Structure, function and reconstruction, Hampton Press, Norfolk). Fullrestoration of function after repair of nerve transection remains anunobtainable ideal because of the fine microstructure of nerves and aninability to achieve precise axon-to-axon coaptation, despite thecurrent state of the art in microsurgical techniques.

Nerve grafting is warranted with nerve ablation but presents severalpractical challenges. Over the years, various nerve graft alternativeshave been explored. Presently viewed as a developing alternative is theapplication of allogenic nerve grafts. While the availability of donorgrafts suffers the difficulties of other organ replacement strategies,the importance of viable cellular elements in nerve grafts may be farless important. Although Schwann cells contribute significantly to theregenerative process, the nerve sheath structure contains the essentialscaffolding and adhesive cues to promote axonal regeneration andsignificant regeneration has been achieved in acellular (e.g.,freeze-killed) nerve grafts (Ide C et al. [1983] Brain Res 288:61-75;Hall S M [1986] Neuropathol Appl Neurobiol 12:401-414; Gulati A K [1988]J Neurosurg 68:117-123; Nadim W et al. [1990] Neuropathol Appl Neurobiol16:411-421). Killing the resident antigen-presenting cells (e.g.,Schwann cells, fibroblasts, endothelial cells, etc.) greatly reduces theimmunogenicity of the graft. Use of acellular nerve grafts greatlyreduces or eliminates the concerns of host-graft immunorejection (EvansP J et al. [1994] Prog Neurobiol 43:187-233; Evans P J et al. [1998]Muscle Nerve 21:1507-1522). These features provide considerable promisefor the use of freeze-killed (acellular) allogenic and xenogenic nervegrafts. On the other hand, the absence of viable cells precludes nervedegeneration and subsequent remodeling which seem to promote theregenerative process (Bedi K S et al. [1992] Eur J Neurosci 4:193-200;Danielsen N et al. [1994] Brain Res 666:250-254).

Laminin is a major growth-promoting component of the basal lamina thatrepresents the adhesive stimulus for successful axonal regeneration(Wang, G Y et al. [1992] Brain Res 570:116-125). However, while normal(uninjured) nerve is rich in laminin, normal nerve remains inhibitive orrefractory to axonal growth. (Langley J N [1904] J Physiol 31:365-391;Brown M C et al. [1994] Eur J Neurosci 6:420-428). This suggests thatthe growth-promoting activity of laminin is suppressed in a normal nerveenvironment and that laminin activity must somehow be revived in nervedegeneration and ensuing regeneration.

Normal peripheral nerve is a poor substratum for axonal growth (Zuo J.et al. [1998] J Neurobiol 34: 41-54; Bedi K S et al. [1992] Eur JNeurosci 4: 193-200). Experimental results indicate that laminin withinnormal nerve basal laminae is not accessible to regenerating axonsprouts (Zuo J. et al. [1998] J Neurosci 18: 5203-5211; Ferguson T A,and D. Muir [2000] Mol Cell Neurosci 16: 157-167; Agius E. et al. [1998]J Neurosci 18: 328-338). Upon injury to the nerve, the severed segment(distal to the injury) undergoes an extensive degenerative process thatinitiates extensive remodeling. In injury-induced nerve degeneration,the severed axons die, their myelin sheath fragments and the resultingdebris are removed by phagocytosis. Despite this degeneration, thesheath structures and basal lamina are preserved. The Schwann cellsproliferate and prepare the nerve for the regrowth of axons. This entireprocess, including the remodeling aspect, is generally referred to asnerve degeneration. It is now clear that nerve injury results inpositive modifications to the distal nerve segment and experiments showthat degenerated nerve has greater axon growth-promoting potential thannormal nerve (Bedi K S et al. [1992] Eur J Neurosci 4: 193-200;Danielsen N J et al. [1994] Brain Res 666: 250-254; Agius E et al.[1998] J Neurosci 18: 328-338). Therefore, the degenerative processappears to involve mechanisms that convert normal nerve from asuppressed state to one that promotes axonal growth (Salonen V J et al.[1987] J Neurocytol 16: 713-720; Danielsen N et al. [1995] Brain Res681: 105-108).

Loss of function associated with nerve injury results from axondisruption. Axons are very thin and fragile and the slightest injury(including compression) can cause a severing response (axotomy). Inaxotomy the axon distal to the lesion dies and degenerates. The leastproblematic injury to a nerve is a crush injury (axonotmesis), wherethere is axotomy but the continuity of the nerve sheaths remains intact.In the case of axonotmesis, axons typically regenerate without surgicalintervention because the basal laminae remain continuous. For severedperipheral nerves to regenerate successfully, axonal sprouts emanatingfrom the proximal nerve stump first must locate and then access Schwanncell basal laminae in the distal nerve segment. This decisiverequirement is thought to contribute to the relatively poor regenerationachieved after nerve transection as compared to crush injury. In nervetransection (neurotmesis) the nerve is partially or fully severed.Transection injuries are those in which both axons and the nerve sheathsare severed, disrupting the continuity of the nerve and the guidancemechanisms required for axon regeneration. Surgical coaptation(neurorrhaphy) to re-establish the continuity of nerve elements of thenerve is essential for regrowth of axons. In addition, axonal regrowthafter nerve transection and repair is further complicated by themisalignment of proximal and distal elements. Even in the instances ofclean transection by a sharp instrument, the entire nerve structure isdisrupted. Swelling and axoplasmic outflow from the cut ends causes amushrooming effect which interferes with accurate coaptation andrealignment of the basal lamina scaffolding. Despite improvements infascicular alignment achieved by microsurgical technique, axon-to-axoncoaptation remains an idealistic goal. Because of the small size ofaxons and the relative preponderance of connective tissues, the majorityof axonal sprouts emerging from the proximal stump after surgicalcoaptation are most likely to first encounter a nonpermissive substratumrich in inhibitory chondroitin sulfate proteoglycan (CSPG). This mayexplain the significant latency and erratic regeneration associated withperipheral nerve transection repair. Evidence indicates that CSPGs bindto and inhibit the growth-promoting activity of laminin and that CSPG isdegraded during the degenerative process after injury. Accordingly, theprocess by which CSPGs are inactivated can explain why regeneration isessential for nerve regeneration. It has recently been found thatperipheral nerve contains abundant CSPG, which inhibits thegrowth-promoting activity of endoneurial laminin (Zuo J et al. [1998a] JNeurobiol 34:41-54). The neurite-inhibiting CSPGs are abundant in theendoneurial tissues surrounding Schwann cell basal laminae and arerapidly upregulated after nerve injury (Braunewell K H et al. [1995a]Eur J Neurosci 7:805-814; Braunewell K H et al. [1995b] Eur J Neurosci7:792-804). Consequently, any misalignment of nerve microstructure(after injury and repair) forces regenerating axonal sprouts tonegotiate nonpermissive tissues which may severely limit their access tobasal laminae in the distal nerve. Recent research supports theconclusion that certain CSPG-degrading enzymes represent a mechanism bywhich the growth-promoting properties of laminin may be restored withindegenerating nerve (Zuo J et al. [1998b] J Neurosci 18:5203-5211;Ferguson T A et al. [2000] Mol Cell Neurosci 16:157-167). In addition,this process can be achieved by the application of CSPG-degradingenzymes at the site of nerve injury and to nerve grafts to improveregeneration (Zuo J et al. [2002] Exp Neurol 176: 221-228; Krekoski C Aet al. [2001] J Neurosci 21: 6206-6213). One such CSPG-degrading enzymethat is particularly effective is chondroitinase ABC, a bacterial enzymethat degrades the disaccharide side-chains of CSPG (Zuo J et al. [1998a]J Neurobiol 34:41-54). Other include specific members of the matrixmetalloproteinase family, MMP-2 and MMP-9, that degrade the core proteinof CSPG (Ferguson T A et al. [2000] Mol Cell Neurosci 16: 157-167).

Although chondroitinase ABC (a glycosaminoglycan lyase) degradeschondroitin sulfate, dermatan sulfate and hyaluronan, its ability toenhance the growth-promoting property of nervous tissue has beenattributed to CSPG degradation (Zuo J et al. [1998] Exp Neurol154:654-662; Ferguson T A et al. [2000] Mol Cell Neurosci 16:157-167).In addition, it has been shown that chondroitinase ABC treatment doesnot disrupt nerve sheath organization or displace laminin from theSchwann cell basal lamina (Krekoski C A et al. [2001] J Neurosci21:6206-6213).

In nerve transection repair models, degradation of inhibitory CSPGremoved a major obstacle to regenerating axonal sprouts and resulted inmore robust and uniform growth into the distal nerve (Krekoski C A etal. [2001] J Neurosci 21:6206-6213).

It has been shown that degenerated nerve has an increased ability tosupport axonal growth (Giannini C et al. [1990] J Neuropathol Exp Neurol49:550-563; Hasan N et al. [1996] J Anat 189:293-302). The effects ofdegeneration are likely due to modifications of the nerve basal laminasince axonal regeneration is also improved into acellular graftsprepared from predegenerated nerve (Danielsen N et al. [1995] Brain Res681:105-108). Throughout the degenerative process, the Schwann cellbasal lamina remains structurally intact.

Animal models have shown that grafts made from nerves that arepredegenerated in vivo are much better at supporting nerve regenerationthan freshly-cut grafts (Danielsen N et al. [1995] Brain Res681:105-108). However, the procedure for creating pre-degenerated nervesin humans is impractical (i.e., nerve injury followed by a period ofsurvival in vivo to allow tissue degeneration).

Peripheral nerve degeneration in vivo results in an increased turnoverof several extracellular matrix molecules which depends on the releaseand activation of proteolytic enzymes by neurons, Schwann cells andinvading macrophages. Modulation of matrix metalloproteinase (MMP)activities after injury implicates MMP-2 and MMP-9 in remodeling of theextracellular matrix during nerve degeneration and regeneration (LaFleur et al. [1996] J Exp Med 184:2311-2326; Kherif et al. [1998]Neuropathol Appl Neurobiol 24:309-319; Ferguson et al. [2000] Mol CellNeurosci 16:157-167). MMP-9 is expressed in the peripheral nerveimmediately after injury and mainly at the site of injury. MMP-9expression correlates with the breakdown of the blood-nerve barrier, theaccumulation of granulocytes and the invasion of macrophages (Shubayevet al. [2000] Brain Res 855:83-89; Siebert et al. [2001] J NeuropatholExp Neurol 60:85-93). Most evidence suggests that hematogenic cellscontribute significantly to the elevation of MMP-9 activity (Taskinen etal. [1997] Acta Neuropathol (Berl) 93:252-259). On the other hand, MMP-2is expressed constitutively by Schwann cells in normal peripheral nerve(Yamada et al. [1995] Acta Neuropathol (Berl) 89:199-203). Several daysafter injury, MMP-2 expression is upregulated and latent enzyme issubstantially converted to its active form (Ferguson et al. [2000] MolCell Neurosci 16:157-167).

In vitro degeneration results in a substantial increase in theneurite-promoting activity of nerve explants. This increase is blockedby the addition of MMP inhibitor, as is the coincidental increase in netgelatinolytic activity (demonstrated by in situ zymography). The rise inneurite-promoting activity occurs rapidly in the cultured nerve explantsand in parallel with the upregulation and activation of MMP-2. Incontrast, the initial effect of in vivo degeneration only suppresses thealready low neurite-promoting activity of normal nerve, during whichtime there is no change in MMP-2 expression or activation in vivo. Theneurite-promoting activity of transected nerve does, however, increaseover time in vivo and this coincides with a burst of MMP-2 expressionand activation (Ferguson and Muir, 2000, Mol Cell Neurosci 16:157-167;Shubayev and Myers, 2000, Brain Res 855:83-89).

In vitro assays indicate that nerve segments predegenerated in vivo havegreater neurite-promoting activity than normal segments of nerve (Bediet al. [1992] Eur J Neurosci 4:193-200; Agius et al. [1998] J Neurosci18:328-338; Ferguson et al. [2000] Mol Cell Neurosci 16:157-167).However, in vivo studies testing predegenerated nerve grafts haveproduced conflicting results, especially when using cellular (live)nerve grafts (Gordon et al. [1979] J Hand Surg [Am] 4:42-47; Danielsenet al. [1994] Brain Res 666:250-254; Hasan et al. [1996] J Anat 189(Pt2):293-302). Nonetheless, predegeneration appears to be particularlyadvantageous for the enhancement of regeneration into acellular grafts(Ochi et al. [1994] Exp Neurol 128:216-225; Danielsen et al. [1995]Brain Res 681:105-108). This indicates that, in degeneration, cellularand molecular mechanisms act to enhance the growth-promoting propertiesof the basal lamina which then retains the ability to stimulate nerveregeneration after the cellular elements have been killed. In vitropredegeneration results in a substantial increase in thegrowth-promoting ability of acellular nerve grafts, that was readilydemonstrated in the present invention's cryoculture and grafting models.Acellular nerve grafting is associated with a substantial latency in theonset of axonal regeneration (Danielsen et al. [1995] Brain Res681:105-108).

Much of the research on nerve explant culture and nerve graftpreservation has focused on the cold storage of nerve segments. Unlikethe efforts to promote finite degeneration of nerve grafts in culture,cold storage methods aim to preserve the nerve in minimal and ischemicconditions that suppress cellular and proteolytic activities. Levi etal. (Levi A et al. [1994] Glia 10:121-131) found that cell viabilitydecreases significantly after 1 week and only a few viable Schwann cellsremained in nerve explants after 3 weeks of cold storage. Subsequently,Lassner et al. (Lassner et al. [1995] J Reconstr Microsurg 11:447-453)reported that culture medium (DMEM, rather than Cold Storage Solution)has a positive effect on maintaining Schwann cell viability and on theregenerative potential of nerve grafts stored in cold ischemicconditions. Although not beneficial for optimizing the growth-promotingpotential of nerve grafts, continued cold storage does further decreasecell viability, immunogenicity and the concerns of immunorejection ofallogenic nerve grafts (Evans et al. [1998] Muscle Nerve 21:1507-1522).For this reason, prolonged cold storage and freeze-killed nerveallografts result in better regeneration that fresh allografts (Evans etal. [1999] Microsurgery 19:115-127).

Accordingly, there remains a need in the art for a low risk adjunctivetherapy to improve the outcome of conventional nerve repair.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns compositions and methods for promotingthe repair of nerve tissue. In a preferred embodiment, the compositionsof the subject invention comprise chondroitin sulfate proteoglycan(CSPG)-degrading enzymes. In one embodiment, a composition of thesubject invention comprises a CSPG-degrading enzyme selected from thegroup consisting of chondroitinase, hyaluronidase, and matrixmetalloproteinase (MMP), or combinations thereof. In a furtherembodiment, a composition of the subject invention comprises aCSPG-degrading enzyme selected from the group consisting ofchondroitinase ABC, chondroitinase A, chondroitinase C, chondroitinaseAC, hyaluronidase, MMP-2, and MMP-9, or combinations thereof.

The present invention also concerns methods to promote the repair ofdamaged nerve tissue in a human or animal. Methods of the presentinvention comprise administering one or more CSPG-degrading enzymes to anerve repair, coaptation, graft, or damaged nerve tissue. The methods ofthe subject invention improve the ability of regenerating axons totraverse the nerve-nerve or nerve-graft interface and potentiates axonalgrowth within the basal lamina scaffold. The degradation of inhibitoryCSPG creates a more permissive nerve substratum and allows axon sproutsgreater access to Schwann cell basal lamina of the nerve, therebyincreasing the number of axons that successfully penetrate damaged nervetissue or implanted nerve grafts. The appropriate routing of the axonsprouts may also be enabled leading to further improvements in recoveryof function.

The present invention also concerns methods of preparing nerve grafts bytreatment with CSP-degrading enzymes. Preferably, the nerve graft(either allogenic or xenogenic) is fresh and not degenerated and istreated with CSPG-degrading enzymes either before or after the nervegraft is frozen. If treated while the cells of the graft are alive, thegraft can be implanted as such or can then be freeze-killed to render itacellular. In one embodiment, the nerve tissue is rendered acellularafter treatment. In a preferred embodiment, the nerve tissue is renderedacellular by freeze-killing.

The present invention also concerns methods of culturing fresh (orbriefly preserved for transport) nerve tissue for subsequentimplantation as a nerve graft into a human or animal. Preferably, thenerve tissue is harvested fresh from human or animal donor and culturedunder physiological conditions that permit the tissue to degenerate andremodel ex vivo, promoting proliferation of Schwann cells within thetissue and activation of the basal lamina by endogenous processes. Inone embodiment, the nerve tissue/graft is rendered acellular afterculturing. In a preferred embodiment, the nerve tissue/graft is renderedacellular by freeze-killing.

The present invention further pertains to methods of providing nervegrafts for implantation into humans or animals. Preferably, thecross-sectional characteristics of the donor graft are similar to thecross-sectional characteristics of the nerve tissue at the implantationsite.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show CSPG neoepitope immunofluorescence ofchondroitinase-treated acellular nerve grafts. Acellular (freeze-killed)rat sciatic nerve segments were treated en bloc with chondroitinase ABCfor 16 h in vitro. FIG. 1A shows neoepitope (chondroitinase-dependent)labeling with Ab1918, demonstrating that en bloc treatment withchondroitinase effectively permeated all nerve compartments and degradedCSPG side-chains. In FIG. 1B, the intensity of Ab1918 immunolabeling wasnot increased by additionally treating sections of the nerve shown inFIG. 1A with chondroitinase, indicating the initial en bloc treatmentwas thorough. In FIG. 1C, the structural integrity of Schwann cell basallaminae in chondroitinase-treated acellular nerve segments was shown bylaminin immunofluorescence. FIG. 1D shows Ab1918 immunolabeling ofchondroitinase-treated acellular interpositional nerve graft after 8days in vivo.

FIG. 2 shows inactivation of inhibitory CSPG by cryoculture bioassays ofacellular nerve segments treated with chondroitinase. Acellular nervesegments were treated en bloc with chondroitinase (“Ch'ase”) or vehiclealone. The nerves were sectioned and then treated additionallypost-treated with chondroitinase or vehicle only. Dissociated chickembryonic DRG neurons were grown on the nerve sections for 24 h andneurite lengths were scored as described in Materials and Methods.Determinations were made by scoring at least 250 neurons in eachcondition. Results are expressed as means (−SEM) and statisticalsignificance comparing the en bloc vehicle and chondroitinase conditionswas determined using Student's t test. * P<0.001.

FIG. 3 shows an assessment of the continuity and GAP-43 immunostainingof interpositional acellular nerve grafts. The continuity of each nervegraft was confirmed by examining the proximal and distal nerve-graftcoaptations in longitudinal section. At the proximal coaptation, GAP-43labeling revealed numerous regenerating axons entering the proximalaspect of the graft. GAP-43 did not label any remnant elements withinthe acellular graft.

FIG. 4 shows axonal regeneration into acellular interpositional nervegrafts after 8 days. Representative series of sections from two animals,each receiving vehicle-treated and chondroitinase-treated grafts. Serialsections taken from the proximal graft (1.2 mm, top) and subsequent 0.56mm intervals were immunolabeled with GAP-43. In each animal receivingbilateral grafts (n=9), axon growth was greater into the acellular grafttreated with chondroitinase than in the vehicle-treated control. Imageswere cropped at the epineurium to approximate the fields scored bydigital image analysis in FIG. 5.

FIG. 5 shows greater accession of regenerating axons intochondroitinase-treated acellular nerve grafts. Serial sections of 8-dayinterpositional nerve grafts (as shown in FIG. 4) were scored forGAP-43-labeled axonal profiles by digital image analysis. Data representthe means (−SEM) of 9 vehicle-treated and 9 chondroitinase-treatedgrafts assessed at the specified distances into the graft (proximal todistal).

FIG. 6 shows axonal regeneration into the initial segment of acellularinterpositional nerve grafts after 4 days. The nerve-graft interface andimmediately proximal region of 4-day acellular grafts were examined.GAP-43-labeled axon profiles were compared at 0.3 mm into the grafts.Data represent the means (−SEM) of 3 vehicle-treated and 3chondroitinase-treated grafts.

FIG. 7 shows the association of axon regeneration and Schwann cellmigration within the grafts. Serial sections of 8-day grafts wereimmunolabeled for GAP-43 (axons) and S-100 (Schwann cells). In proximalregions of the chondroitinase-treated grafts, Schwann cells were mostoften found in close association with regenerating axons. Occasionalclusters of axons were observed without comigrating Schwann cells(arrow). At more distal points in the grafts, axons were often foundwithout accompanying Schwann cells. Few isolated Schwann cells wereintensely immunolabeled for S-100 in the more distal regions of thegrafts, which contained mostly faint S-100 staining associated withfreeze-killed Schwann cell remnants.

FIGS. 8A and 8B show axon and Schwann cell growth at the distal graftcoaptation. Serial longitudinal sections of 8-day chondroitinase-treatedgrafts and distal nerve stumps were immunolabeled for GAP-43 (axons), asshown in FIG. 8A, and for 5-100 (Schwann cells), as shown in FIG. 8B. InFIG. 8A, axons (small arrows) approach, traverse the distal coaptation,and grow diffusely within the host distal stump. In FIG. 8B, S-100labeled Schwann cells are abundant in the distal host stumps, yet few ifany invade the distal aspect of the grafts (which contains faint S-100immunostaining associated with freeze-killed Schwann cell remnants).

FIGS. 9A and 9B show human nerves stained for CSPG neoepitope andlaminin, respectively. These results show that, although the grossstructure of human nerve is more complex than rat nerve, the basallamina which supports axon regeneration is mainly similar and themolecular components that regulate growth (CSPG and laminin) areabundant. FIG. 9A also demonstrates that, by virtue of the neoepitopelabeling, CSPG side chains were effectively degraded in human nervesegments treated with chondroitinase.

FIG. 10 shows inactivation of inhibitory CSPG by cryoculture assaysusing human nerve segments. Human nerves were treated withchondroitinase and then assayed for neurite-promoting activity.Dissociated chick DRG neurons were grown on the sections for 24 h andneurite lengths were scored. Results are expressed as means (−SEM).Statistical significance (P<0.001) comparing vehicle-treated andchondroitinase-treated conditions was found using Student's t test.

FIG. 11 shows greater growth of axons into chondroitinase-treatedacellular nerve grafts in a human-to-rat xenograft model. Human nervefascicles (of similar diameter to the rat sciatic nerve) were graftedinto a gap made in the rat sciatic nerve. Serial sections of the 8-dayinterpositional nerve xenografts were scored for GAP-43-labeled axonalprofiles by digital image analysis. Data represent the means (−SEM) of 2vehicle-treated and 2 chondroitinase-treated grafts assessed at thespecified distances into the graft (proximal to distal).

FIGS. 12A-12D show degradation of CSPG in the injured sciatic nerve by asingle injection of chondroitinase ABC. Two injury models were examined,bilateral nerve transection and repair (FIGS. 12A, 12B, and 12D) andbilateral nerve crush (FIG. 12C). At the time of injury the rightsciatic nerve was injected with chondroitinase ABC (1 U in 2 μl) at asite 2 mm distal to the site of nerve injury. Four days after nervetransection and repair, CSPG-neoepitope immunostaining was intensethroughout the endoneurium and nerve sheaths at the coaptation site(FIG. 12A) (note sutures in the epineurium) and throughout thecross-sectional area of the nerve several mm both distal (FIG. 12B) andproximal (not shown) to the coaptation. As shown in FIG. 12C, similarresults were obtained in crush-injured nerves which were examined 2 daysafter chondroitinase injection. The extent of CSPG degradation by invivo injection of chondroitinase was examined by CSPG-neoepitopeimmunolabeling of nerves treated a second time with chondroitinase afterthe tissue was sectioned, as shown in FIG. 12D. The staining intensityobserved in serial sections was not noticeably different after thesecond application (compare FIG. 12B and FIG. 12D), indicating thesingle in vivo injection of chondroitinase effectively degraded CSPG inthe surrounding extracellular matrix.

FIGS. 13A and 13B show treatment with chondroitinase ABC did not alteraxonal regeneration after nerve crush injury. Adult rats receivedbilateral sciatic nerve crush and one nerve was injected withchondroitinase ABC and the contralateral nerve received vehicle alone.Nerves were removed two days after injury and regenerating axons werelabeled by GAP-43 immunocytochemistry. Regenerated axon profilesimmediately distal to the nerve crush in two representative animals(each receiving vehicle and chondroitinase injections) are shown in FIG.13A. As shown in FIG. 13B, GAP-43-immunolabeled axons were scored inserial sections of the distal nerves. There was no significantdifference in axon regeneration in the chondroitinase-treated (Ch'ase)nerves compared to the vehicle-treated control nerves. Data representthe means (±SEM) of 6 chondroitinase-treated and 6 vehicle-treatednerves assessed at 0.56-mm intervals into the distal nerves.

FIGS. 14A and 14B show treatment with chondroitinase ABC markedlyenhanced axon regeneration after nerve transection and neurorrhaphicrepair. Adult rats received bilateral nerve transection and end-to-endrepair. One nerve was injected with chondroitinase ABC and thecontralateral nerve received vehicle alone. Nerves were removed fourdays after injury and regenerating axons were labeled by GAP-43immunocytochemistry. Regenerated axon profiles immediately distal to thenerve coaptation in two representative animals (each receiving vehicleand chondroitinase injections) are shown in FIG. 14A.GAP-43-immunolabeled axons were scored in serial sections of the distalnerves as shown in FIG. 14B. Axon regeneration was significantly greaterin the chondroitinase-treated (Ch'ase) nerves compared to thevehicle-treated controls. Data represent the means (±SEM) of 7chondroitinase-treated and 7 vehicle-treated nerves assessed at 0.56-mmintervals into the distal nerves.

FIGS. 15A and 15B show the cryoculture assay of nerve explant cultures.As shown in FIG. 15A, freshly excised rat sciatic nerve explants werecultured for 1, 2, 4, and 7 days in DMEM/N2 containing 0, 2, or 10%fetal bovine serum. As shown in FIG. 15B, nerve explants were culturedfor 2 days in DMEM/N2 containing 2% serum (Culture Standard) without andwith the addition of GM6001 (MMP inhibitor). The nerves were thencryosectioned and embryonic DRG neurons were seeded onto the tissuesections in DMEM/N2 containing NGF. After 24 hours, DRG neurons wereimmunostained for GAP-43 and neuritic growth was measured by digitalphotomicroscopy and image analysis. The control condition was normalnerve (0 days in culture). Data represent the mean neurite lengths(±SEM) of >250 neurons scored in each condition from at least 4 separatenerve explant cultures tested in 2 or more separate experiments.

FIG. 16 shows the zymographic analysis of nerve explant cultures. Nerveexplants were cultured for 0 (Control, C), 1, 2, 4, and 7 days inDMEM/N2 containing 2% serum. The nerves were then extracted and analyzedby gelatin-overlay electrophoresis. Zymography reveals both proform andactivated gelatinases which appear as clear bands within the stainedgel. Control nerve contained predominantly pro-MMP-2 and trace amountsof activated MMP-2. There was a progressive increase in MMP-2 contentand a rapid conversion to the activated form within the nerve explantscultured for 2 days or longer. MMP-9 was negligible in the control andearly explants whereas a trace amount was detected at 4 and 7 days. Themolecular masses indicate the positions of recombinant human pro-MMP-9(92 kD), activated MMP-9 (84 kD), pro-MMP-2 (72 kD) and activated MMP-2(66 kD).

FIGS. 17A-17F show the localization of net gelatinolytic activity innerve segments by in situ zymography. Tissue sections of control nerve(FIG. 17A and FIG. 17B) and cultured nerve explants (2-day, 2% serum)(FIG. 17C and FIG. 17D) were overlaid with quenched, fluorescein-labeledgelatin, which is converted to fluorescent peptides by gelatinolyticactivity within tissues. Constitutive gelatinolytic activity wasdetected in normal nerve (FIG. 17A) which, at higher magnification (FIG.17B), was associated with Schwann cells. As shown in FIGS. 17C and 17D,gelatinolytic activity was more intense and diffuse throughout theendoneurium in the cultured nerves. As shown in FIGS. 17E and 17F,gelatinolytic activity in nerves cultured in the presence of GM6001 wasmarkedly decreased.

FIGS. 18A-18D show immunoexpression of MMP-2 and MMP-9 in cultured nerveexplants. As shown in FIG. 18A, MMP-2 immunolabeling of culture nerves(2-day, 2% serum) was intense within Schwann cells and the surroundingbasal laminae (inset). In FIG. 18B, S-100 immunolabeling showed therepositioning of an expanded population of Schwann cells within thenerve. As shown in FIG. 18C, MMP-9 immunolabeling was virtually absentwithin the nerve fascicles, except for a rare cellular profile. Somecells in the surrounding epineurium were labeled for MMP-9. In FIG. 18DOX42 labeling showed macrophages scattered throughout the epineurium andrarely within the nerve fascicles of cultured nerves.

FIGS. 19A-19D show Wallerian degeneration in cultured nerve explants.The degenerative changes observed in the nerve segments cultured for 2days were reminiscent of the initial phases of Wallerian degenerationseen in vivo. In FIG. 19A, neurofilament immunolabeling showed thecompact and contiguous formation of axons in normal nerve compared tothe annular and fragmented axons found in cultured nerve explants(2-day, 2% serum) as shown in FIG. 19B (FIGS. 19A and 19B insets,longitudinal sections). As shown in FIG. 19C, immunolabeling for lamininindicated that basal laminae were structurally intact and that lamininexpression was upregulated in Schwann cells (inset). As shown in FIG.19D, the degeneration of axons and the extrusion of myelin by Schwanncells was especially evident in semi-thin sections stained withtoluidine blue. Degenerative processes resulting in further myelindegeneration (collapse and condensation) and phagocytotic removal werenot observed in the 2-d cultured nerve segments as shown in the inset ofFIG. 19D.

FIGS. 20A and 20B show axonal regeneration within acellular nerve graftspredegenerated in vitro. Normal and cultured (2-day, 2% serum) nervegrafts were freeze-killed, trimmed to 10 mm in length and used asinterpositional grafts for the repair of transected sciatic nerves. Hostrats received bilateral grafts, one normal (uncultured) and onepredegenerated (cultured). Axonal regeneration was assessed after 8 daysby scoring GAP-43-immunopositive profiles in transverse sections. InFIG. 20A, representative sections of control and predegenerated graftsfrom two animals are shown. Sections show the axonal regeneration at 1.5mm into the grafts. Pixel values of the immunofluorescent images wereinverted. As shown in FIG. 20B, quantitative analysis was performed atmeasured distances within the grafts. Data represent the means (±SEM) of6 nerves in each condition.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention provides compositions and methods for promotingthe repair of nerve tissue. The compositions and methods of the subjectinvention can be employed to restore the continuity of nerve interruptedby disease, traumatic events or surgical procedures. The compositionsand methods of the subject invention promote repair of nerve tissue byincreasing the number of axons that successfully penetrate damaged nervetissue or implanted nerve grafts, resulting in greater functionalrecovery.

In a preferred embodiment, the compositions of the subject inventioncomprise chondroitin sulfate proteoglycan (CSPG)-degrading enzymes. Inone embodiment, a composition of the subject invention comprises aCSPG-degrading enzyme selected from the group consisting ofchondroitinase, hyaluronidase, and matrix metalloproteinase (MMP), orcombinations thereof. In a further embodiment, a composition of thesubject invention comprises a CSPG-degrading enzyme selected from thegroup consisting of chondroitinase ABC, chondroitinase A, chondroitinaseC, chondroitinase AC, hyaluronidase, MMP-2, and MMP-9, or combinationsthereof.

The CSPG-degrading enzymes can be human, animal, or bacterial in origin,naturally occurring or recombinant. As used herein, the term“CSPG-degrading enzymes” is also intended to include biologically activefragments and variants of such enzymes, e.g., that retain a substantialamount of their CSPG-degradative activity. The compositions of thesubject invention can include an appropriate pharmaceutical carrier. Thesubject invention further concerns nerve tissue treated with one or moreCSPG-degrading enzymes.

In addition to one or more CSPG-degrading enzymes, the compositions ofthe subject invention can further comprise biologically orpharmacologically active molecules, such as growth factors. Such growthfactors include, but are not limited to, nerve growth factor (NGF),fibroblast growth factors (FGF-1 and 2), epidermal growth factor (EGF),ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor(BDNF), neurotrophin-3, -4, and -5 (NT-3, -4, and -5), insulin-likegrowth factor-I and -II (IGF-I, II), transforming growth factor (TGF),glial growth factor-2 (GGF-2), vascular endothelial growth factor(VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), andlymphocyte infiltrating factor/cholinergic differentiating factor(LIF/CDF). Such molecules can be obtained naturally or by recombinantDNA techniques. Fragments or variants of such molecules that retaintheir biological or pharmacological activities can also be used.

The present invention also concerns methods to promote the repair ofdamaged nerve tissue in a human or animal. Methods of the presentinvention comprise administering one or more CSPG-degrading enzymes to anerve graft or damaged nerve tissue. The methods of the subjectinvention improve the ability of regenerating axons to traverse thenerve-nerve and nerve-graft interface and potentiates axonal growthwithin its basal lamina scaffold. The degradation of inhibitory CSPGcreates a more permissive nerve substratum and allows axon sproutsgreater access to Schwann cell basal lamina of the nerve, therebyincreasing the number of axons that successfully penetrate damaged nervetissue or implanted nerve grafts.

Application of CSPG-Degrading Enzymes to Damaged Nerve.

In one embodiment, the CSPG-degrading enzymes are applied to damagednerve, the site of nerve damage or the site of nerve damage repair. In apreferred embodiment, the CSPG-degrading enzymes are applied to the siteof primary nerve repair involving coaptation of severed or trimmed nerve(i.e., end-to-end nerve coaptation). The damage to the nerve canrepresent a nerve transection (neurotmesis), wherein the nerve ispartially or fully severed or a small region damaged and surgicallyremoved, and epineurial coaptation (neurorrhaphy) is the primary methodof repairing the damaged nerve. For example, the compositions andmethods of the subject invention can be used to promote repair of nervedamage that involves a disruption in the continuity of at least one ofthe nerve sheaths of the damaged nerve, such as the basal lamina,perineurium, or epineurium. Preferably, the surgical repair attempts torealign nerve elements.

In a specific embodiment, the damage to the nerve represents a nervecrush injury (axonotmesis) or more extreme damage, where there isaxotomy but the continuity of the sheath remains intact or is somewhatcompromised. In the case of axonotmesis, axons typically regeneratewithout surgical intervention.

In some cases, a segment of the nerve is diseased, irreparably damagedor obliterated and is surgically removed. Repair may involveimplantation of a graft or prosthesis to bridge the gap. The implant maybe natural (e.g., nerve or vascular graft), a natural derivative (e.g.,biopolymer tube) or synthetic conduit (silicone tube). These areconnected to the cut nerve ends. In a specific embodiment, theCSPG-degrading enzymes are applied at the connection sites, at either orboth ends. For example, the CSPG-degrading enzymes can be applied to oneor both points of host-graft interface on an interpositional graft. TheCSPG-degrading enzymes can be applied before, during, or after surgicalrepair of the damaged nerve tissue or implantation of the graft withinthe recipient.

Application of CSPG-Degrading Enzymes to Nerve Grafts.

In one embodiment, the CSPG-degrading enzymes are applied to a nervegraft. When the CSPG-degrading enzymes are applied to a nerve graft, theentire graft can be treated. The CSPG-degrading enzymes can be appliedto the entire nerve graft, en bloc. This application is a pretreatmentor incubation prior to implantation and may or may not involveprocedures to remove the applied enzyme. The en bloc treatment can beapplied to living (fresh) or previously frozen nerve grafts. The en bloctreatment does not preclude, but may be used in conjunction with,additional application of CSPG-degrading enzymes at the site ofcoaptation with host nerve.

According to the methods of the subject invention, the CSPG-degradingenzyme can be applied to the nerve graft or damaged nerve tissue, orboth. The CSPG-degrading enzyme can be applied to a nerve graft before,during, or after implantation. The CSPG-degrading enzyme can be appliedto any portion of the graft, such as the end or ends to be joined to thestump of the damaged nerve. If the CSPG-degrading enzyme is applied tothe damaged nerve, the enzyme can be applied to any area of the damagednerve that promotes repair of the damaged nerve, such as at the site ofdamage or adjacent to the site of damage. The CSPG-degrading enzymes canbe placed in a culture medium for application to the nerve graft. Theculture medium can be undefined medium, defined medium, or definedmedium supplemented with serum for example. The subject invention alsoincludes storage solutions for storage of nerve grafts prior toimplantation. The storage solution contains a culture medium, asindicated above, and at least one CSPG-degrading enzyme. The storagesolution can also include a tissue adhesive, such as fibrin glue. Thestorage solution can also include other biologically active agents, suchas the growth factors listed above.

As used herein, the term “graft” refers to any tissue intended forimplantation within a human or animal. Various types of graft areencompassed within the subject invention, such as autografts, syngrafts,allografts, and xenografts. The size (e.g., length and diameter) of thegraft is not critical to the subject invention. For example, the lengthof the nerve graft can be from about 1 centimeter to about 10centimeters, or over about 10 centimeters. The diameter of the nervegraft can match that of any injured nerve or part of a nerve, as needed.The nerve graft can be a structurally complete segment of nerve tobridge a gap along the length of the recipient's nerve or to replace thedistal end, i.e., for end-to-end grafting. Alternatively, the nervegraft can be a partial nerve segment, or eccentrically-shaped (e.g., anerve flap), and intended to reconstruct a lacerated nerve that has somestructural disruption, but retains its physical continuity.

Optionally, the CSPG-degrading enzyme can be applied to the injurednerve or nerve graft in conjunction with a tissue adhesive, such as abiological glue. Preferably, the biological glue is a fibrin-containingadhesive, such as fibrin glue, fibrin sealant, or platelet gel.Biological glues are well known in the surgical art (Suri A et al.[2002] Neurol. India 50:23-26; Alibai E et al. [1999] Irn J. Med. Sci.24(3&4):92-97; Sames M et al. [1997] Physiol. Res. 46(4):303-306;Jackson M et al. [1996] Blood Coag. Fibrinolysis 7:737-746; Fasol R etal. [1994] J. Thorac. Cardiovasc. Surg. 107:1432-1439). As used herein,the terms “fibrin glue”, “fibrin sealant”, and “fibrin tissue adhesive”are used interchangeably to refer to a group of formulations containingfibrinogen and thrombin, which lead to the formation of a fibrin clot atthe site of application. The tissue adhesive can be appliedsimultaneously or consecutively with the CSPG-degrading enzyme. Thetissue adhesive can be applied to the injured nerve and/or nerve graftwithin the same formulation as the CSPG-degrading enzyme, or in aseparate formulation. Preferably, the adhesive will not containsubstances such as laminin that will attract the growth of axons fromthe remaining nerve structure or contain substrates or inhibitors forthe applied enzyme(s) that will compete with or inhibit activity of theenzyme(s).

The CSPG-degrading enzymes used in the subject invention can be appliedto the nerve graft or damaged nerve tissue by various means and in avariety of formulations. As used herein, the terms “applied”,“administered”, “contacted”, and “treated” are used interchangeably. Forexample, the CSPG-degrading enzymes can be applied to the nerve graft ordamaged nerve tissue topically (e.g., drop-wise), or administered byinjection. Topical application or local administration by injection arepreferred for greater control. Further, the CSPG-degrading enzymes, orcompositions containing such enzymes, are preferably applied as aliquid, flowable, formulation. The CSPG-degrading enzyme or enzymes canalso be adsorbed onto a porous substance, or formulated into anointment, salve, gel, cream, or foam, for example.

The subject invention also includes kits for promoting repair of damagednerve tissue. The kits of the invention include a first compartmentcontaining at least one CSPG-degrading enzyme and a second compartmentcontaining a tissue adhesive, such as those described herein.Optionally, the kits can include a third compartment for mixing theCSPG-degrading enzyme or enzymes and the tissue adhesive. The kits canbe used for repair of damaged nerve tissue directly, or indirectly, vianerve graft. The kit can include packaging of various materials known inthe art, such as plastic, glass, and/or paper products.

Pharmaceutical Compositions.

One or more CSPG-degrading enzymes can be incorporated into apharmaceutical composition suitable for administration to a patient,e.g., a human or animal. Such compositions typically comprise at leastone CSPG-degrading enzyme and a pharmaceutically acceptable carrier. Asused herein, the term “pharmaceutically acceptable carrier” is intendedto include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Supplementary active compounds can also beincorporated into the compositions. Preferably, the pharmaceuticalcompositions include at least one CSPG-degrading enzyme and a tissueadhesive, such as fibrin glue.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sourceswhich are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Science (Martin E W [1995]Easton Pennsylvania, Mack Publishing Company, 19^(th) ed.) describesformulations which can be used in connection with the subject invention.Formulations suitable for parenteral administration include, forexample, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules, vials,and disposable syringes made of glass or plastic, and may be stored in afreeze dried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that, inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion. The pharmaceutical compositions can be included in acontainer, pack, or dispenser, together with instructions foradministration.

The CSPG-degrading enzymes can be formulated in a carrier appropriatefor the mode of administration, e.g., saline or aqueous buffer. TheCSPG-degrading enzymes can also be contained within, or associated with,a controlled release formulation. Such materials include, but are notlimited to, biodegradable matrices and particles, such as liposomes,lipospheres, or vesicles. The controlled release formulation can be abiodegradable polymeric matrices. The CSPG-degrading enzymes can also beapplied as a gel or film, or contained within a synthetic graft orimplant.

The CSPG-degrading enzymes can be prepared with carriers that willprotect the enzymes against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Preferably, the carrier is biodegradable and/orbioresorbable. Biodegradable, biocompatible polymers can be utilized inthe controlled release formulation, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid.

The controlled release formulation can be particulate in nature (e.g.,of micro- or nano-size scale), such as a sphere or capsule. The particlecan have a core containing one or more CSPG-degrading enzymes, which isencapsulated by an outer layer or shell. The outer shell can bedegradable by the encapsulated CSPG-degrading enzyme (such that theshell is degraded from within). For example, the shell can be at leastpartially composed of hyaluronan, such that when the hyaluronan withinthe shell is degraded (partially or completely) by the encapsulatedCSPG-degrading enzyme or enzymes, the CSPG-degrading enzyme or enzymesare released. Alternatively, the shell can be degraded by anotherdegrading agent that is either exogenously applied or that is presentwithin the in vivo environment (such that the shell is degraded fromwithout).

U.S. Pat. No. 5,320,837 describes controlled release preparationsobtained by reacting an enzyme having an amino group, such ashyaluronidase or chondroitinase, with a copolymer of maleic anhydrideand a copolymerizable polyalkylene glycol ether. The reaction product issoluble in water and/or organic solvent and capable of slowly releasingthe enzyme upon hydrolysis.

U.S. Pat. No. 4,933,185 describes a controlled release system fordelivery of a biologically active substance consisting of an enzyme(such as hyaluronidase) encapsulated within a microcapsule having a coreformed of a polymer, such as an ionically cross-linked polysaccharide,which is specifically degraded by the enzyme and a rate controllingskin. The integrity of the skin is lost when the core is degraded,causing a sudden release of the biologically active substance from thecapsule. The controlled release system in the '185 patent can beutilized to deliver a CSPG-degrading enzyme or enzymes. For example, theCSPG-degrading enzyme or enzymes can function as the biologically activesubstance, or the core degrading enzyme, or both.

The controlled release formulation can provide an initial exposure ofthe CSPG-degrading enzyme or enzymes, followed by one or more delayedexposures following a specific period of time. Alternatively, thecontrolled release formulation can cause a single delayed release of theCSPG-degrading enzyme or enzymes. Alternatively, the continuous releaseformulation can allow for continuous release of the CSPG-degradingenzyme or enzymes. Optionally, the continuous release of theCSPG-degrading enzyme or enzymes can be in conjunction with one or morepulsed releases.

The carrier of the CSPG-degrading enzymes, such as an implant, can be ofa size and shape appropriate for the particular application. Thus, thecarrier can be of a desired volume and in a desired shape, designed indue consideration of the region of the living body at which the carrieris put to use. Examples of shapes include, but are not limited to, acylinder, a semicylinder, or a ring. The carrier can be a pad, a wrap, asheet, a bar, or a thread that is contacted with the injured nerve ornerve graft. Preferably, the carrier does not have shape edges orcorners that may irritate or otherwise stimulate the surrounding tissueof the living body.

The amount of CSPG-degrading enzyme or enzymes released from the carrierand the duration of release can be controlled within appropriate ranges.The carrier can be fixed or secured to the graft or injured nerve or totissue adjacent to the graft or injured nerve. The carrier cancontinuously release the CSPG-degrading enzyme or enzymes at the nerveinjury site over a period of time, such as, for example, 24 hours tothree months.

Depending upon the particular carrier utilized, the CSPG-degradingenzyme or enzymes can be contained within, coated, or otherwiseassociated with the carrier during or after its manufacture. Forexample, the CSPG-degrading enzyme or enzymes can be associated with acommercial product.

The carrier can also function to deliver other biologically activeagents, such as cells (e.g., Schwann cells) or growth factors, with theCSPG-degrading enzymes. The cells delivered by the carrier can bederived from the patient, or from another source of the same species ora different species. The cells delivered by the carrier can begenetically modified to produce a biologically active agent.

In one embodiment, the carrier is a surgical cuff, such as thosedescribed in U.S. Pat. Nos. 4,602,624, 5,487,756, and published U.S.Patent Application No. 2002/0071828, which can be implanted closelyadjacent to the nerve graft or injured nerve (e.g., at the site ofdamage). The cuff of the subject invention includes a sleeve to beapplied to the nerve graft or damaged nerve tissue. The sleeve can be avariety of shapes. For example, the sleeve can be a tubular prosthesisor wrap that at least partially or fully encircles the damaged nerveand/or nerve graft and may include any device that is compatible withthe intended use of joining the ends of an injured nerve either directlyor indirectly through a nerve graft, using a cuffing technique, torestore nerve continuity. If the cuff is tubular in shape, the cuff canoptionally include a longitudinal slit with abutting first and secondedges for ease of application to a nerve graft or damaged nerve. Forexample, the first and second abutting edges of the longitudinal slitcan be in separable contact with one another, permitting the separationof the abutting edges of the slit, exposing the lumen of the tubularsleeve. The damaged nerve and/or nerve graft can then be inserted intothe lumen, allowing the abutting edges of the longitudinal slit toreturn to being in separable contact with one another holding thedamaged nerve and/or nerve graft together and available for exposure toCSPG-degrading enzymes.

Optionally, the surgical cuff can be secured to the nerve usingconventional suture techniques or a tissue adhesive, such as abiological glue that can be applied to the nervous system, or othermeans. Preferably, the biological glue is a glue containing fibrin, suchas BIOCOLLE (BIOTRANSFUSION), CRTS, (Lille), ISSUCOL (IMMUNO AG, ViennaAustria), and the like. The cuff can be a rigid support or, for example,a self-curling sheet. The self-curling sheet can automatically encirclethe damaged nerve an/or nerve graft when contacted to the respectivetissue. The cuff can be permeable, impermeable, or semi-permeable.Optionally, the cuff can include a means for electrically stimulatingthe nerve graft or damaged nerve and/or a means for recording nerveelectrical activity within the nerve graft or damaged nerve, such asthat described in U.S. Pat. No. 5,487,756. Preferably, theCSPG-degrading enzyme or enzymes are released or otherwise operate fromthe inner surface of the cuff, i.e., that surface facing the nerve graftor damaged nerve.

The surgical cuff can provide the CSPG-degrading enzyme or enzymes tothe nerve graft or damaged nerve via a delivery system, such as areservoir or an expression system, such as the adenovirus constructsdescribed in published U.S. Patent Application No. 2002/0071828.Expression systems for chondroitin lyase enzymes are known in the art,some of which are described in U.S. Pat. Nos. 6,054,569; 6,093,563;published U.S. Patent Application No. 2001/0034043; and Tralec, A. L.[2000] Appl. Environ. Microbiol. 66:29-35.

The surgical cuff can be composed of a variety of synthetic material(s),such as silicone, PAN/PVC, PVFD, polytetrafluoroethylene (PTFE) fibersor acrylic copolymers. In a specific embodiment of the invention, theuse of a cuff consisting of or based on biomaterials, such as inparticular cross-linked collagen, bone powder, carbohydrate-basedpolymers, polyglycolic/polylactic acid derivatives, hyaluronic acidesters, or chalk-based supports, is preferred. Preferably, collagen orsilicone is used within the framework of the present invention. It maybe collagen of, for example, human, bovine or murine origin. Morepreferably, a cuff consisting of a bilayer of type I or III or IV,advantageously IV/IVox, collagen, or of silicone, is used. There may bementioned, by way of a specific example, a SILASTIC cuff (DOW-CORNING),consisting of silicone. Moreover, the cuff may have advantageously atubular shape, of cylindrical or angular section. The diameter of thecuff can be adjusted by persons skilled in the art according to thedesired applications. In particular, for stimulating the regeneration ofa peripheral nerve, a relatively small diameter, from 0.05 to 15 mm, canbe used. More preferably, the inner diameter of the cuff is between 0.5and 10 mm. For spinal cord regeneration applications, cuffs with alarger inner diameter can be chosen. In particular, for theseapplications, the cuffs used have an inner diameter which may be as highas 15 to 20 mm, depending on the relevant nerve section. For bridging aroot avulsed at the level of the brachial plexus, the diameter of thecuff advantageously corresponds to the diameter of the root. The lengthof the cuff is generally determined by the size of the loss of substanceto be compensated for. Cuffs with a length of between 0.5 and 5 cm canbe used. Preferably, the length of the cuff remains less than 5 cm,losses of substance greater than 5 cm being less frequent.

The CSPG-degrading enzymes can be applied to the nerve graft or damagednerve tissue in various concentrations, but are preferably applied in aconcentrated form. Ideal concentrations will vary with nerve size andenzyme. For example, chondroitinase can be applied in a concentrationranging from about 10 units/mL to about 1000 units/mL. Preferably, thechondroitinase is applied to the nerve graft or damaged nerve tissue ata concentration range from about 100 units/mL to about 500 units/mL.MMPs can be applied in a concentration ranging from about 0.1 μg/mL toabout 100 μg/mL. Preferably, the MMP is applied in a concentrationranging from about 10 μg/mL to about 50 μg/mL.

As indicated above, according to the methods of the subject invention,the CSPG-degrading enzyme or enzymes can be administered to a nervegraft or injured nerve tissue in conjunction with a biologically activemolecule, such as a growth factor. Other biologically active agents thatcan be administered with the CSPG-degrading enzyme or enzyme includegenetically-modified or non-genetically modified cells. Thus, thecompositions of the subject invention can include such cells. The cellscan be non-stem cells (mature and/or specialized cells, or theirprecursors or progenitors) or stem cells. Thus, the administered cellscan range in plasticity from totipotent or pluripotent stem cells (e.g.,adult or embryonic), precursor or progenitor cells, to highlyspecialized or mature cells, such as those of the central or peripheralnervous system (e.g., Schwann cells).

Stem cells can be obtained from a variety of sources, including fetaltissue, adult tissue, cord cell blood, peripheral blood, bone marrow,and brain, for example. Stem cells and non-stem cells (e.g., specializedor mature cells, and precursor or progenitor cells) can bedifferentiated and/or genetically modified. Methods and markers commonlyused to identify stem cells and to characterize differentiated celltypes are described in the scientific literature (e.g., Stem Cells:Scientific Progress and Future Research Directions, Appendix E1-E5,report prepared by the National Institutes of Health, June, 2001). Thelist of adult tissues reported to contain stem cells is growing andincludes bone marrow, peripheral blood, brain, spinal cord, dental pulp,blood vessels, skeletal muscle, epithelia of the skin and digestivesystem, cornea, retina, liver, and pancreas.

According to the methods of the subject invention, genetically modifiedhosts, such as recombinant cells, can be administered to the nerve graftor damaged nerve tissue. The hosts can be genetically modified toproduce one or more CSPG-degrading enzymes. Preferably, theCSPG-degrading enzyme is secreted from the recombinant cell. Forexample, expression systems for chondroitin lyase enzymes are known inthe art, some of which are described in U.S. Pat. Nos. 6,054,569;6,093,563; published U.S. Patent Application No. 2001/0034043; andTralec, A. L. [2000] Appl. Environ. Microbiol. 66:29-35. Optionally, therecombinant host is genetically modified to recombinantly produce otherbiologically active agents, in addition to the CSPG-degrading enzyme.

Nucleic acid molecules encoding one or more CSPG-degrading enzymes canbe inserted into vectors and used as gene therapy vectors. Gene therapyvectors can be delivered to a patient by, for example, intravenousinjection, local administration, or by stereotactic injection. Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease carrier in which the gene delivery vehicle is imbedded orotherwise associated. In addition, the pharmaceutical preparation caninclude a therapeutically effective amount of cells which recombinantlyproduce the CSPG-degrading enzyme.

The various methods employed in the genetic modification of host cellsare well known in the art and are described, for example, in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, second edition,volumes 1-3, Cold Spring Harbor Laboratory, New York, and Gloves, D. M.(1985) DNA Cloning, Vol. I: A Practical Approach, IRL Press, Oxford.Thus, it is within the skill of those in the genetic engineering art toextract DNA from its source, perform restriction enzyme digestions,electrophorese DNA fragments, tail and anneal plasmid and insert DNA,ligate DNA, transform cells, e.g., prokaryotic and eukaryotic cells,prepare plasmid DNA, electrophorese proteins, and sequence DNA.

To reduce immunogenicity, nerve grafts used in the subject invention canbe made acellular by a variety of methods known to those of ordinaryskill in the art. For example, the nerve tissue can be made acellular byfreeze-killing, as described in the Materials and Methods section, or bychemical extraction with detergents (Sondell M et al. [1998] Brain Res795:44-54). The nerve grafts can be rendered acellular before, during,or after application of one or more CSPG-degrading enzymes.

In Vitro Nerve Culture.

The present invention also concerns methods of culturing nerve tissuefor implantation into a human or animal. The culture methods of thesubject invention involve “predegenerating” the nerve tissue in vitro,which, following engraftment, improves the ability of regenerating axonsto traverse the interface between the graft and host nerve tissue.Without being bound by theory, the culturing methods of the subjectinvention allow the living nerve cells to express CSPG-degrading enzymesand promote Schwann cell proliferation, as would occur naturally in vivoduring the remodeling process of nerve degeneration.

The method of in vitro culture involves culturing the nerve tissue underconditions that permit the nerve tissue to grow in vitro and increasethe neurite-promoting activity of the nerve tissue when subsequentlyimplanted as a graft. The increase in neurite-promoting activity can beas determined by an in vitro neurite outgrowth assay of the nervetissue, such as the cryoculture bioassay described herein.

Alternatively, an in vivo neurite outgrowth assay of the nerve tissuecould also be utilized. Methods for assaying neurite outgrowth are knownin the art and typically involve qualitatively or quantitativelydetermining the extent of neurite outgrowth on a solid support, such asa microplate or microscope slide. Standard fluorescence an be utilized.

The methods of the subject invention can comprise isolating nerve tissuefrom a human or animal and culturing the nerve tissue for a short periodof time in vitro, ranging from about 24 hours to about 96 hours. Longerincubations in vitro can result in deterioration and loss ofgrowth-promoting properties. Preferably, the nerve tissue is culturedfrom about 24 hours to about 72 hours. More preferably, the nerve tissueis cultured for about 48 hours.

The nerve tissue can be cultured at a temperature within a range ofabout 10° C. to about 37° C. Preferably, the nerve tissue is culturedwithin a range of about 30° C. to about 37° C. More preferably, thenerve tissue is cultured at about 37° C.

The nerve tissue can be cultured in defined medium or mediumsupplemented with serum. The defined medium can be, for example, N2medium or Dulbecco's Modified Eagle Medium (DMEM). If mediumsupplemented with serum is used, the serum can be human or animal, suchas fetal bovine serum. Preferably, the nerve tissue is cultured indefined medium. In one embodiment, the nerve tissue is a nerve graftthat is rendered acellular after culturing and prior to implantationwithin a host. In a preferred embodiment, the nerve graft is renderedacellular by freeze-killing. While no exogenous enzymes are necessary tocarry out the culture methods of the subject invention, the methods canfurther comprise contacting the nerve tissue with one or moreCSPG-degrading enzymes.

The present invention further pertains to methods of providing nervegrafts for implantation into humans or animals. Preferably, thecross-sectional characteristics of the nerve graft are similar to thecross-sectional characteristics of the host nerve tissue at theimplantation site, e.g., the host's proximal and distal nerve stump. Inone embodiment, the method of the subject invention comprises generatingdigital image data of the nerve stump cross section within a potentialhost (i.e., graft recipient), analyzing the image data to definecoordinate locations of nerve elements and their diameter to produce arecipient template, and comparing the recipient template data to donortemplate data that can be stored in memory. The donor template datarepresents the digital image data from a “bank” of stored nerve grafts.The stored nerve graft with the highest degree of structural elementalignment with the recipient's nerve stump can then be selected forimplantation within the recipient. The relevant parameters include thediameter, thickness, and/or spatial arrangement (i.e., boundaries) ofone or more of the structural elements, which include, but are notlimited to, epineurium, fascicular groups, fascicles, myelin sheath, andaxons. Therefore, alignment between the nerve graft and the host nervecan be maximized. Preferably, the nerve graft selected is one with asimilar cross-sectional arrangement of fascicular groups and axons.

U.S. Pat. No. 5,231,580 describes a variety of methods for determiningthe characteristics of nerve. The generation of the digital image datacan be achieved using methods and devices well known in the art, such asa digital camera. Analysis of the image data and comparison of therecipient template data to the stored donor template data can beachieved, for example, through an algorithm capable of image scanning,analysis, and pattern recognition. To select the closest match betweennerve graft and recipient nerve, threshold values of similarity can beestablished.

The methods and compositions of the subject invention are applicable tonerve tissue of both the central nervous system (CNS) and peripheralnervous system (PNS). For example, nerve grafts of the subject inventioncan be used as interpositional nerve grafts in the PNS or as bridges inthe brain and spinal cord and any extensions thereof.

The CSPG-degrading enzymes used in the subject invention can be obtainedfrom a variety of sources, including organisms that produce the enzymenaturally or organisms that produce (or overproduce) the enzyme throughgenetic modification (producing a recombinant enzyme). For example, theCSPG-degrading enzymes can be obtained from bacterial sources, includingthose that naturally produce the enzyme, or those that have beengenetically modified to produce (or overproduce) the enzyme.CSPG-degrading enzymes can also be obtained from mammalian sources,including those mammals that naturally produce the enzyme or thosemammals that have been genetically modified to produce (or overproduce)the enzyme. Alternatively, the CSPG-degrading enzyme can be chemicallysynthesized.

As used herein, the “proximal” part is intended to mean the part of theaxon that remains in continuity with the neuron cell bodies or the partof the nerve containing these axons. The “distal” part is intended tomean the part of the axon that becomes disconnected from the neuron cellbody or the part of the nerve containing these disconnected axons.

In the case of a peripheral nerve lesion, its proximal part is thatwhich is connected to the ganglia or spinal cord. The distal part of theperipheral nerve is intended to mean the peripheral-most part of thenerve that is connected to the motor endplate (neuromuscular junction)or sensory organs. In the case of a lesion of the spinal cord, theproximal part is that which is in contact with nuclei or more anterior.The distal part is intended to mean that part which extends to aterminal synapse.

The terms “treating” or “treatment”, as used herein, refer to reductionor alleviation of at least one adverse effect or symptom associated withthe particular nerve damage suffered by the patient.

As used herein, the term “stem cell” is an unspecialized cell that iscapable of replicating or self renewal, and developing into specializedcells of a variety of cell types. The product of a stem cell undergoingdivision is at least one additional stem cell that has the samecapabilities of the originating cell. For example, under appropriateconditions, a hematopoietic stem cell can produce a second generationstem cell and a neuron. Stem cells include embryonic stem cells (e.g.,those stem cells originating from the inner cells mass of theblastocyst) and adult stem cells (which can be found throughout the moremature animal, including humans). As used herein, stem cells areintended to include those stem cells found in animals that have maturedbeyond the embryonic stage (e.g., fetus, infant, adolescent, juvenile,adult, etc.).

As used herein, the term “progenitor cell” (also known as a “precursorcell”) is unspecialized or has partial characteristics of a specializedcell that is capable of undergoing cell division and yielding twospecialized cells. For example, a myeloid progenitor/precursor cell canundergo cell division to yield two specialized cells (a neutrophil and ared blood cell).

As used herein, the term “co-administration” and variations thereofrefers to the administration of two or more agents simultaneously (inone or more preparations), or consecutively.

As used herein, the term “combination” includes sub-combinations. Forexample, a combination of the CSPG-degrading enzymes chondroitinase ABC,chondroitinase A, chondroitinase C, chondroitinase AC, hyaluronidase,MMP-2, and MMP-9, would include subcombinations of chondroitinase ABCand MMP-2, for example.

As used herein, the term “biological activity” or “biologically active”is intended to refer to the activity associated with the particularagent, molecule, compound, etc. For example, the biological activityexhibited by CSPG-degrading chondroitinases is degradation of CSPG.Preferably, the CSPG-degrading activity includes cleavage or lysis ofchondroitin-4-sulfate, chondroitin-6-sulfate, or bothchondroitin-4-sulfate and chondroitin-6-sulfate. Hence, biologicallyactive fragments and variants of specific CSPG-degrading enzymes exhibitCSPG-degrading activity, as well. Likewise, biologically activefragments of growth factors, such as fibroblast growth factor-1, exhibitthe biological activity normally associated with that growth factor.

The term “genetic modification” as used herein refers to the stable ortransient alteration of the genotype of a cell of the subject inventionby intentional introduction of exogenous nucleic acids by any meansknown in the art (including for example, direct transmission of apolynucleotide sequence from a cell or virus particle, transmission ofinfective virus particles, and transmission by any knownpolynucleotide-bearing substance) resulting in a permanent or temporaryalteration of genotype. The nucleic acids may be synthetic, or naturallyderived, and may contain genes, portions of genes, or other usefulpolynucleotides. The term “genetic modification” is not intended toinclude naturally occurring alterations such as that which occursthrough natural viral activity, natural genetic recombination, or thelike.

Various vectors can be utilized to carry out genetic modificationaccording to the subject invention. The vectors can be vaccine,replication, or amplification vectors. In some embodiments of thisaspect of the invention, the polynucleotides are operably associatedwith regulatory elements capable of causing the expression of thepolynucleotide sequences. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations of theaforementioned vector sources, such as those derived from plasmid andbacteriophage genetic elements (e.g., cosmids and phagemids).

As indicated above, vectors utilized to carry out genetic modificationcan also comprise elements necessary to provide for the expressionand/or the secretion of a polypeptide, such as a CSPG-degrading enzyme,or a biologically active fragment or variant thereof, encoded by thenucleotide sequences of the invention in a given host cell. The vectorcan contain one or more elements selected from the group consisting of apromoter, signals for initiation of translation, signals for terminationof translation, and appropriate regions for regulation of transcription.In certain embodiments, the vectors can be stably maintained in the hostcell and can, optionally, contain signal sequences directing thesecretion of translated protein. Other embodiments provide vectors thatare not stable in transformed host cells. Vectors can integrate into thehost genome or be autonomously-replicating vectors.

In a specific embodiment, the vector comprises a promoter operablylinked to a protein or peptide-encoding nucleic acid sequence, one ormore origins of replication, and, optionally, one or more selectablemarkers (e.g., an antibiotic resistance gene). Non-limiting exemplaryvectors for the expression of the polypeptides of the invention includepBr-type vectors, pET-type plasmid vectors (PROMEGA), pBAD plasmidvectors (INVITROGEN) or those provided in the examples below.Furthermore, vectors according to the invention are useful fortransforming host cells for the cloning or expression of the nucleotidesequences of the invention.

Promoters which may be used to control expression include, but are notlimited to, the CMV promoter, the SV40 early promoter region (Bernoistand Chambon [1981] Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al. [1980] Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al. [1981]Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al. [1982] Nature 296:39-42);prokaryotic vectors containing promoters such as the β-lactamasepromoter (Villa-Kamaroff et al. [1978] Proc. Natl. Acad. Sci. USA75:3727-3731), or the tac promoter (DeBoer et al. [1983] Proc. Natl.Acad Sci. USA 80:21-25); see also, “Useful Proteins from RecombinantBacteria” in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al. [1983] Nature 303:209-213) or the cauliflowermosaic virus 35S RNA promoter (Gardner et al. [1981] Nucl. Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebiphosphate carboxylase (Herrera-Estrella et al. [1984] Nature310:115-120); promoter elements from yeast or fungi such as the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, and/or the alkaline phosphatase promoter.

The subject invention also provides for the use of “homologous” or“modified” nucleotide sequences. Modified nucleic acid sequences will beunderstood to mean any nucleotide sequence obtained by mutagenesisaccording to techniques well known to persons skilled in the art, andexhibiting modifications in relation to the normal sequences. Forexample, mutations in the regulatory and/or promoter sequences for theexpression of a polypeptide that result in a modification of the levelof expression of a polypeptide according to the invention provide for a“modified nucleotide sequence”. Likewise, substitutions, deletions, oradditions of nucleic acid to the polynucleotides of the inventionprovide for “homologous” or “modified” nucleotide sequences. In variousembodiments, “homologous” or “modified” nucleic acid sequences havesubstantially the same biological or serological activity as the native(naturally occurring) CSPG-degrading enzyme. A “homologous” or“modified” nucleotide sequence will also be understood to mean a splicevariant of the polynucleotides of the instant invention or anynucleotide sequence encoding a “modified polypeptide” as defined below.

A homologous nucleotide sequence, for the purposes of the presentinvention, encompasses a nucleotide sequence having a percentageidentity with the bases of the nucleotide sequences of between at least(or at least about) 20.00% to 99.99% (inclusive). The aforementionedrange of percent identity is to be taken as including, and providingwritten description and support for, any fractional percentage, inintervals of 0.01%, between 20.00% and 99.99%. These percentages arepurely statistical and differences between two nucleic acid sequencescan be distributed randomly and over the entire sequence length.

In various embodiments, homologous sequences exhibiting a percentageidentity with the bases of the nucleotide sequences used in the presentinvention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identitywith the polynucleotide sequence encoding the CSPG-degrading enzyme.

Both protein and nucleic acid sequence homologies may be evaluated usingany of the variety of sequence comparison algorithms and programs knownin the art. Such algorithms and programs include, but are by no meanslimited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson andLipman [1988] Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul etal. [1990] J Mol. Biol. 215(3):403-410; Thompson et al. [1994] NucleicAcids Res. 22(2):4673-4680; Higgins et al. [1996] Methods Enzymol.266:383-402; Altschul et al. [1990] J. Mol. Biol. 215(3):403-410;Altschul et al. [1993] Nature Genetics 3:266-272).

Cells or tissue administered to a patient according to the methods ofthe subject invention can be derived from humans or other mammals,including non-human primates, rodents, and porcines, for example.Specific examples of source species include, but are not limited to,humans, non-human primates (e.g., apes, chimpanzees, orangutans,monkeys); domesticated animals (pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as bovines, buffalo, bison, horses,donkey, swine, sheep, and goats; exotic animals typically found in zoos,such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, giantpandas, hyena, seals, sea lions, elephant seals, porpoises, dolphins,and whales.

Likewise, mammalian species which benefit from the disclosed methods oftreatment include, and are not limited to, humans, non-human primates(e.g., apes, chimpanzees, orangutans, monkeys); domesticated animals(e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamesepot-bellied pigs, rabbits, and ferrets; domesticated farm animals suchas bovines, buffalo, bison, horses, donkey, swine, sheep, and goats;exotic animals typically found in zoos, such as bear, lions, tigers,panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes,sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears,kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions, elephantseals, otters, porpoises, dolphins, and whales.

All patents, patent applications, provisional patent applications, andpublications referred to or cited herein are incorporated by referencein their entirety, as are co-filed U.S. patent application Ser. No.10/218,316 (UF-336XC1) “Materials and Methods for Nerve Repair”; andU.S. patent application Ser. No. 10/218,864 (UF-336XC3) “Materials andMethods for Nerve Grafting, Selection of Nerve Grafts, and In VitroNerve Tissue Culture”, including all figures, tables, drawings,nucleotide sequences, and amino acid sequences, to the extent they arenot inconsistent with the explicit teachings of this specification.

MATERIALS AND METHODS

Surgical Procedures for Nerve Transection and Nerve Crush Experiments.

All surgical procedures were performed according to Institutional AnimalCare and Use Committee (IACUC) approved protocols. Young adult SPRAGUEDAWLEY rats (HARLAN Indianapolis, Ind.) were deeply anesthetized withxylazine (15 mg/kg, i.m.) followed by ketamine-HCl (110 mg/kg, i.p.).Six animals received bilateral nerve crush injuries. Sciatic nerves wereexposed and then crushed with firm pressure for thirty seconds with aDUMONT #5 forceps at a site 4 mm distal to the tendon of the internalobdurator. The crush site was marked with an epineurial suture. In aseparate set of experiments, eight rats received bilateral sciatic nervetransection injuries using serrated scissors. The proximal and distalstumps were coated by epineurial neurorrhaphy using 9-0 ETHILON sutures.Fibrin glue (fibrinogen and thrombin) was then applied to stabilize theunion. In both injury models, the right sciatic nerves were injected2-mm distal to the injury with chondroitinase ABC (1 U in 2 μl)(high-purity, protease-free; SIGMA CHEMICAL CO., St. Louis, Miss.). Leftsciatic nerves (with the same injury as the right side) were injectedwith vehicle alone (0.1% bovine serum albumin in PBS). Muscle incisionswere sutured and the skin closed with metal clips. After recovery fromthe anesthetic, animals were returned to standard housing. Two days (forcrush injury) and four days (for transection injury) after surgery,nerves were removed under anesthesia and fixed as described below. Oneof the eight animals receiving nerve transection and repair was excludedbecause loss of continuity in one nerve occurred during convalescence.

Preparation of Acellular Nerve Grafts Treated with Chondroitinase.

Adult (180-200 g) female SPRAGUE DAWLEY rats (HARLAN, Indianapolis,Ind.) were used as nerve donors and recipient hosts. Donor rats wereanesthetized with halothane and decapitated. Sciatic nerves were exposedthrough a gluteal muscle-splitting incision and isolated free ofunderlying fascia. A 15-mm nerve segment was excised rostral to thebifurcation into common peroneal and tibial nerves. The segments wererinsed with cold sterile Ringer's solution, stabilized by pinning theends to a thin plastic support, and transferred to a cryogenic vial. Thevials were submerged in liquid nitrogen for 2 minutes and thentransferred to a 37° C. water bath for 2 minutes. This freeze/thaw cyclewas repeated, yielding acellular nerve grafts that were then stored inliquid nitrogen. On the day before grafting, the nerve grafts werewarmed to room temperature and incubated in 100 μl phosphate bufferedsaline pH 7.4 (PBS) containing 2 units/ml chondroitinase ABC (SIGMA, St.Louis, Mo.) or in PBS (vehicle) only for 16 hours at 37° C. The graftswere rinsed twice with Ringer's and kept on ice prior to use. Thechondroitinase ABC preparation was highly purified and stated by themanufacturer to be essentially free of protease activity.

Interpositional Nerve Grafting for Chondroitinase Experiments.

Twelve rats received bilateral acellular nerve grafts, onechondroitinase-treated and one vehicle-treated graft. Host rats weredeeply anesthetized using xylazine (15 mg/kg, i.m.) and ketamine (110mg/kg, i.p.). The sciatic nerve was exposed and supported by a plasticinsert placed between the nerve and underlying tissue. The region ofnerve halfway between the sciatic notch and bifurcation was first coatedwith fibrin glue. Using serrated scissors, a 2.5-mm segment of hostnerve was excised and replaced with a freshly trimmed 10-mm acellularnerve graft. The graft was coapted to the host nerve stumps byepineurial neurorrhaphy using one 9-0 ETHILON suture at each end. Fibringlue was then applied to stabilize the coaptations which, in combinationwith the initial fibrin coating, also reduced protrusion of nerveelements (endoneurial mushrooming) (Menovsky T et al. [1999]Neurosurgery 44:224-226). The muscle was closed with 4-0 sutures and theskin with wound clips. After recovery from the anesthetic, animals werereturned to standard housing.

Nine rats were terminated at 8 days and four at 4 days after grafting.Animals were deeply anesthetized and decapitated. The graft and 3 mm ofproximal and distal host nerve were removed, immersed in 4%paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4° C.The specimens were equilibrated with PBS and immersed in 30%sucrose/phosphate buffer for 2 days at 4° C. Using a dissectingmicroscope and the epineurial sutures as landmarks, each specimen wassubdivided into 3 segments representing a) the proximal nerve-graftinterface, b) the main graft and c) the distal nerve-graft interface.The specimens were embedded and cryosectioned. Longitudinal sectionswere taken through the nerve-graft interfaces to examine the continuityof the coaptations.

The main grafts were sectioned serially on the transverse plane inrecorded measure to assess the extent of axonal growth by microscopy.Regenerating axons were labeled by GAP-43 immunofluorescence (see below)in sections of the grafts at 0.56 mm intervals. Epifluorescentphotomicrographs were acquired using a SPOT Digital Camera System(DIAGNOSTIC INSTRUMENTS, INC., Sterling Heights, Mich.) and AXIOVERT 10microscope (CARL ZEISS, Thornwood, N.Y.). GAP-43-positive axon profileswere scored using IMAGE-PRO PLUS software (MEDIA CYBERNETICS, SilverSprings, Md.).

Nerve Explant Culture for Predegeneration Experiments.

Adult (180-200 gm) female SPRAGUE DAWLEY rats (HARLAN, Indianapolis,Ind.) were used as nerve donors and graft recipients. This project wasreviewed and approved by the Institutional Animal Care and UseCommittee. Donor rats were deeply anesthetized with isofluorane anddecapitated. Sciatic nerves were exposed through a glutealmuscle-splitting incision and isolated free of underlying fascia. A15-mm nerve segment was excised rostral to the bifurcation into commonperoneal and tibial nerves. The segments were rinsed with sterileRinger's solution and stabilized by pinning the ends to a thin plasticsupport. The nerve explants were cultured for 1, 2, 4 and 7 days inDULBECCO'S modified EAGLES' medium containing N2 supplements (DMEM/N2)or DMEM/N2 supplemented with 2% or 10% fetal bovine serum (FBS) (ATLANTABIOLOGICALS, Atlanta, Ga.). As specified, some explants were cultured inthe presence of the MMP inhibitor, GM6001 (50 μM) (Grobelny et al.[1992] Biochem., 31:7152-7154). The cultured nerves were washedthoroughly in DMEM and then transferred to sealed tubes. The tubes wereimmersed in liquid nitrogen for 2 min and then thawed in a 37° C. waterbath for 5 min. This freeze-thaw cycle was repeated twice, yieldingfreeze-killed (acellular) nerve segments. Freshly excised nerves(uncultured controls) were freeze-killed using the same procedure. Theacellular nerve segments were then a) embedded for cryosectioning foruse in cryoculture assays or b) stored in liquid nitrogen (for up to 2weeks) for biochemical analysis and for use as interpositional nervegrafts. Nerve explants prepared for histological examinations were fixedwith aldehydes and freeze-killing was omitted.

Nerve degeneration in vivo was accomplished by a single transection ofthe sciatic nerve near the pelvis. The proximal stump was displaced andligated to preclude axonal growth. The leg muscles and skin were closedand the transected nerve was allowed to degenerate in situ for 2 or 7days.

Immunocytochemistry.

Axonal regeneration was assessed by GAP-43 immunofluorescence anddigital image analysis. Tissue sections mounted on slides were washedwith PBS and then treated with 0.5% Triton X-100 in PBS for 10 min. Thesections were treated with blocking buffer (10% serum in PBS+0.1% TritonX-100) and then incubated overnight at 4° C. with primary antibodies(diluted in blocking buffer). Bound antibodies were labeled with swineanti-rabbit immunoglobulins (DAKO CORPORATION, Carpinteria, Calif.) orgoat anti-mouse immunoglobulins (Sigma) FITC-conjugated secondaryantibodies for 1 h at room temperature in darkness. The anti-mousesecondary antibody was preadsorbed with rat serum prior to use. Thesections were washed, postfixed with 4% paraformaldehyde in PBS, rinsed,and coverslipped in fluorescent mounting media. Affinity-purified rabbitanti-GAP-43 peptide antibody was produced using known methods (FergusonT A et al. [2000] Mol Cell Neurosci, 16:157-167) and was used at 2μg/ml. Polyclonal antibody 1918 (CHEMICON INTERNATIONAL, Temecula,Calif.) (1:1000) binds only to the unsaturated disaccharide unit thatremains attached to the linkage region of CSPG core protein exposed bydigestion with chondroitinase ABC (Bertolotto A et al. [1986] J NeurolSci 73:233-244). Polyclonal anti-EHS laminin antibody (Sigma) (1:1000)was used to label basal laminae. Polyclonal anti-S-100 antiserum (Dako)(1:500) was used to label Schwann cells. Dark-field images were invertedand optimized for printing in PHOTOSHOP (ADOBE SYSTEMS INC., San Jose,Calif.).

Cryoculture Bioassay.

Cryoculture is a neurite outgrowth assay in which neurons are cultureddirectly on fresh/frozen nerve sections and was performed as describedpreviously (Ferguson T A et al. [2000] Mol Cell Neurosci, 16:157-167).Briefly, chondroitinase- and vehicle-treated nerve segments weresectioned at 20 μm, mounted on sterile, aminopropyltriethoxysilane(APTS)-coated coverslips and stored at −20° C. until used. Whereindicated, sections were treated with chondroitinase ABC (0.1 unit/ml)or vehicle (50 mM Tris-HCl, pH 8.0), containing 50 mM NaCl) for 2 h at37° C. Purified dorsal root ganglionic (DRG) neurons from day 8 chickembryos were seeded directly on the nerve sections in a defined N2medium (Bottenstein J E et al. [1980] Exp Cell Res 125:183-190)containing 10 ng/ml nerve growth factor. Cryoculture assays wereterminated after 24 h of incubation by fixation with 100% methanol.Neuritic growth by DRG neurons was accessed by GAP-43 immunofluorescentlabeling. Epifluorescent photomicrographs were acquired as described fortissue sections. Neurite lengths were measured directly using IMAGE-PROPLUS software (MEDIA CYBERNETICS, Silver Springs, Md.). At least 250neurons with neurites greater than one cell body (˜15 μm) were scoredfor each condition in each experiment.

Gel Zymography for Nerve Predegeneration Experiments.

Nerve segments were placed in ice-cold extraction buffer (50 mMTris-HCl, pH 7.6, containing 1% Triton X-100, 200 mM NaCl, and 10 mMCaCl₂) and homogenized by probe sonication (15 sec). The samples wereagitated for 30 min at 4° C. and the soluble fraction collected bycentrifugation (12,000 g, 20 min). Total protein content of the solublefractions was determined using the BRADFORD REAGENT (BIO-RADLABORATORIES, Hercules, Calif.). Bovine serum albumin dissolved inextraction buffer was used as a protein standard. The extracts weresolubilized in non-reducing Laemmli sample buffer without heating andelectrophoresed at 4° C. on 10% SDS-polyacrylamide gels containing 1.5mg/ml porcine gelatin. The gels were briefly rinsed in water and thenwashed in 2.5% Triton X-100 three times over 45 min. The Triton wasremoved with three 5-min water washes and the zymographic gels weredeveloped for 21 h in incubation buffer (50 mM Tris-HCl, pH 8.0, 5 mMCaCl₂, 0.02% sodium azide). Gels were fixed and stained with 0.05%Coomassie brilliant blue. Protein bands with gelatinolytic activityappeared as a clear lysis zones within the blue background of thegelatin gel. Comigration of gelatinolytic bands was compared with latentand activated forms of recombinant human MMP-2 and MMP-9, as well asprestained molecular weight standards (BIO-RAD). Digitalphotomicrographs were acquired and densitometry of gelatinolytic bandswas performed using IMAGE-PRO PLUS software.

In Situ Zymography for Nerve Predegeneration Experiments.

Cryosections (10 μm) of unfixed normal and cultured nerves were mountedon slides and overlaid with reaction buffer (50 mM Tris-HCl, 150 mMNaCl, 5 mM CaCl₂, 0.2 mM sodium azide, pH 7.6) containing 20 μg/ml ofintramolecularly quenched, fluorescein-labeled gelatin substrate(MOLECULAR PROBES INC., Eugene, Oreg.) (Oh et al., 1999). In the controlcondition, the MMP inhibitor EDTA (30 mM) was included in the reactionbuffer. After incubation for 24 h at 37° C., the sections were rinsedwith PBS and fixed with 4% paraformaldehyde in phosphate buffer. Thesections were rinsed with water and mounted using Citifluor.Fluorescein-gelatin peptides generated by gelatinolytic activity in thetissue sections were observed and photographed by epifluorescencemicroscopy.

Interpositional Nerve Grafting for Predegeneration Experiments.

Six rats were given bilateral acellular nerve grafts, one normal(uncultured) and one predegenerated in vitro (cultured for 2 d in 2%serum). Host rats were deeply anesthetized using xylazine (15 mg/kg,i.m.) and ketamine (110 mg/kg, i.p.). The sciatic nerve was exposed andsupported by a plastic insert placed between the nerve and underlyingtissue. The region of the nerve halfway between the sciatic notch andbifurcation was first coated with fibrin glue. A 2.5-mm segment of thehost nerve was excised using serrated scissors. The graft was thawed andfreshly trimmed to 10 mm with a scalpel blade. The graft was coapted tothe host nerve stumps by epineurial neurorrhaphy using one 9-0 Ethilonsuture at each end. Fibrin glue was then applied to stabilize thecoaptations that, in combination with the initial fibrin coating appliedto the host nerve, reduced protrusion of nerve elements (endoneurialmushrooming) (Menovsky and Bartels, 1999, Neurosurgery, 44:224-225,discussion pp. 225-226). The muscle was closed with 4-0 sutures and theskin was closed with wound clips. After recovery from the anesthetic,animals were returned to standard housing. Eight days after grafting thehost rats were deeply anesthetized and decapitated. The graft and 3 mmof proximal and distal host nerve were removed and immersed in 4%paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, overnight at 4° C.The specimens were equilibrated with PBS and immersed in 30% sucrose inphosphate buffer for 2 d at 4° C. The specimens were embedded andcryosectioned on the transverse plane in a recorded measure.Regenerating axons within the grafts were labeled by GAP-43immunofluorescence (see below). Epifluorescent photomicrographs wereacquired and GAP-43-positive axon profiles were scored using IMAGE-PROPLUS software.

Immunofluorescent Labeling for Nerve Predegeneration Experiments.

Fixed tissue sections were treated with 0.5% Triton X-100 in PBS for 10min. Non-specific antibody binding was blocked by pretreatment with PBScontaining 0.1% Triton X-100 and 10% normal serum (Blocking buffer).Primary antibodies were diluted in Blocking buffer and applied overnightat 4° C. Bound primary antibodies were labeled with swine anti-rabbitimmunoglobulins (DAKO, Carpinteria, Calif.) or goat anti-mouseimmunoglobulins (Sigma) conjugated with fluorescein or rhodamine for 1hour at room temperature in darkness. The anti-mouse secondary antibodywas pre-adsorbed with rat serum prior to use. Neurite length(cryoculture) and axonal regeneration (grafting) were assessed byimmunolabeling with polyclonal anti-GAP-43 IgG (2 μg/ml) (Ferguson andMuir, 2000, Mol Cell Neurosci, 16:157-167) (NB300-143; NOVUS BIOLOGICAL,Littleton, Colo.). Other primary antibodies included: polyclonalanti-MMP-2 IgG (4 μg/ml) (MMP2/475; Muir, 1995); polyclonal anti-MMP-9IgG (4 μg/ml) (AB19047; CHEMICON, Temecula, Calif.); polyclonalanti-S-100 antiserum (1:500) (DAKO) and; polyclonal OX42 antiserum(1:500) (SEROTEK, Raleigh, N.C.); and monoclonal anti-neurofilament IgG(4 μg/ml) (NAP4; Harris et al., 1993). In some instances, epifluorescentphotomicrographs were inverted and contrast-enhanced for printing inPHOTOSHOP (ADOBE SYSTEMS, San Jose, Calif.).

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Degradation of CSPG by Treatment of Acellular Nerve Segmentswith Chondroitinase

The purpose of this experiment was to determine if chondroitinasetreatment effectively degraded CSPG throughout intact segments ofacellular nerves. Segments of rat sciatic nerve (1.5 cm in length) weremade acellular by repeated freeze-thaw cycles and then bathed en bloc ina chondroitinase ABC solution for 16 hours. CSPG degradation within thechondroitinase pretreated nerves was examined by immunolabeling withneoepitope antibody Ab1918. This antibody binds to an epitope created onthe core protein after lysis of the chondroitin sulfate chains bychondroitinase ABC (Bertolotto et al. [1986] J. Neurol Sci, 73:233-244).Ab1918 immunostaining was intense throughout the entire pretreated nervesegment, as shown in FIG. 1A. Furthermore, the intensity of Ab1918immunostaining was not increased by an additional post-treatment of thesections with chondroitinase, as shown in FIG. 1B. Ab1918immunoreactivity was absent in acellular nerves not exposed tochondroitinase (not shown). These findings indicate that the en blocchondroitinase treatment effectively permeated all nerve compartmentsand thoroughly degraded CSPG side-chains.

In normal nerve, CSPG and laminin are mainly colocalized in the nervesheaths and basement membranes, including Schwann cell basal laminae(Zuo et al. [1998a] J. Neurobiol., 34:41-54). Their distributions wereunchanged after repeated freeze-thaw and there was no indication at thelight microscopic level that en bloc chondroitinase treatment alteredECM structures, as shown in FIGS. 1A and 1C). The integrity ofchondroitinase-treated acellular nerve segments was an importantconsideration for their subsequent use as nerve regeneration grafts.Accordingly, the structural integrity of the pretreated nerve segmentsafter nerve grafting was also examined. The intensity and distributionof Ab1918 immunoreactivity (in regions of the grafts not infiltrated byhost cells) was unchanged after 8 days in vivo, indicating the primarystructure of Schwann cell basal laminae remained intact, as shown inFIG. 1D. Taken together, these results demonstrate that en blocchondroitinase treatment of acellular nerve grafts effectively degradedCSPG without compromising the basal lamina scaffold or dislocating itslaminin content.

Example 2—Inactivation of Inhibitory CSPG by Treatment of AcellularNerve Segments with Chondroitinase

Inactivation of inhibitory CSPG in chondroitinase-treated acellularnerve was determined by cryoculture bioassay. Embryonic chick DRGneurons were seeded onto sections of prepared nerve segments and theneurite-promoting activity was assessed by scoring neurite growth.Results are shown in FIG. 2. On sections of acellular nerve pretreateden bloc with vehicle only the average neurite length was 49 μm. Neuritegrowth on acellular nerve pretreated en bloc with chondroitinaseaveraged 96 μm, representing a 95% increase compared to the controlcondition. To determine if the en bloc chondroitinase treatment wasthorough, cryoculture assays were performed on nerve tissues treatedwith chondroitinase after sectioning (post-treatment). As expected, theneurite-promoting activity of acellular nerve treated en bloc withvehicle only was increased significantly (86%) by post-treatment withchondroitinase. In contrast, chondroitinase post-treatment had only aslight additive effect on sections from en bloc chondroitinase-treatednerve grafts.

These results indicate that inhibitory CSPG was effectively degraded andinactivated by bathing segments of acellular nerve grafts in smallamounts of chondroitinase ABC. In addition, en bloc chondroitinasetreatment effectively deinhibited the nerve grafts without disruptingthe laminin-associated, neurite-promoting potential of the basal laminascaffold. The latter point was strengthened by the observation that,like in cryoculture assays of normal and degenerated nerve (Ferguson andMuir, 2000, Mol Cell Neurosci, 16:157-167), neurite growth on sectionsof chondroitinase-treated acellular nerve grafts occurred in strictassociation with Schwann cell basal laminae.

Example 3—Nerve Regeneration is Enhanced by Chondroitinase Treatment ofAcellular Nerve Grafts

The following experiments tested the hypothesis that chondroitinasetreatment improves nerve regeneration through acellular nerve syngrafts.As described in Example 2, acellular sciatic nerve segments were treateden bloc with vehicle or chondroitinase ABC. Ten-mm interpositional nervegrafts were joined to the host nerve by epineurial neurorrhaphyreinforced with fibrin glue. Each of nine host rats received bilateralgrafts, one vehicle-treated and one chondroitinase-treated graft.Regeneration was initially examined after 8 days. First, the proximaland distal nerve-graft coaptations were examined in longitudinal sectionto assess the alignment of the surgical coaptation, as shown in FIG. 3.All of the grafts were in continuity and thus were included in thesubsequent analysis. Scoring of regeneration was based onGAP-43-immunolabeling which intensely stained growing axons. Axon andSchwann cell remnants within the freeze-killed grafts wereimmunonegative for GAP-43 and host Schwann cells were only very faintlystained (at an intensity below the threshold used for digital scoring).Axonal growth was assessed at specified spatial intervals within thegraft by scoring GAP-43-immunopositive profiles in transverse sections.Some axonal ingrowth was observed in all grafts, as shown in FIG. 4.However, the growth into chondroitinase-treated grafts was markedlygreater and more widely distributed than in control grafts. Quantitativeresults are shown in FIG. 5.

The average number of axons (GAP-43-immunopositive profiles) enteringchondroitinase-treated grafts was on average more than three-foldgreater than in control grafts. While the axons entering the controlgrafts were always restricted and most often clustered centrally, theinitial growth into chondroitinase-treated grafts was more widelydispersed and especially abundant at the proximal end. These findingsindicate that the success of axonal penetration into acellular nervegrafts was markedly improved by pretreatment of the grafts withchondroitinase. However, a similar number of axons was consistentlyobserved at the distal ends of grafts in both conditions. This suggestedthat axonal penetration into the control grafts occurred early and thenwas temporally restricted while axons continued to penetratechondroitinase-treated grafts throughout the 8-day period.

To determine if the latency of axonal growth into acellular grafts wasreduced by chondroitinase treatment, the same analysis was performed on4-day grafts except that the most proximal aspects of the grafts wereexamined and scored in transverse section as well. Although only 3animals receiving bilateral grafts were examined, the results wereconsistent with those observed for 8-day grafts. Moreover, at the mostproximal aspect of the graft (0.3 mm from the host-graft interface)axonal penetration was on average five-fold greater inchondroitinase-treated grafts, as shown in FIG. 6. From these results,it can be concluded that chondroitinase treatment decreases the latencyand significantly improves the accession of axonal regeneration intoacellular nerve grafts.

Example 4—Axon Regeneration within Basal Lamina Tubes ofChondroitinase-Treated Grafts

Because the success of nerve regeneration depends on the growth of axonswithin the laminin-rich, basal lamina tubes, it was determined whetherthe association of axonal growth with basal laminae was altered bychondroitinase-treatment of acellular grafts. Transverse sections of8-day grafts were double-labeled for GAP-43 and laminin. Lamininlabeling was intense and basal laminae appeared similarly intactthroughout control and chondroitinase-treated grafts. Despite repeatedfreeze-thaw, enzyme treatment, surgical manipulation and 8 days in vivo,the extracellular matrix scaffold appeared structurally intact. MultipleGAP-43-labeled axons (or neurites) were evident within individual basallaminae and the vast majority of these were observed in closeassociation with the lumenal surface of the tubes. A similar and minornumber of neurites with ambiguous apposition were observed in controland treated grafts. By and large, the propensity of axons to grow withinbasal laminae was unaltered by chondroitinase treatment of acellularnerve grafts.

Example 5—Axonal Growth and Schwann Cell Migration intoChondroitinase-Treated Grafts

Serial sections of the 8-day grafts were immunolabeled for S-100 andGAP-43 to examine the migration of Schwann cells in respect to axongrowth. The grafts contained two distinct patterns of S-100 staining;intense staining was associated with live, host-derived Schwann cellsand faint staining with freeze-killed Schwann cell remnants. Thedescriptions that follow refer to the intensely stained (live) Schwanncell profiles, unless otherwise indicated. In proximal regions of thegrafts the distributions of Schwann cells and axons mainly coincided, asshown in FIG. 7. Occasional clusters of axons were found without anyapparent Schwann cell association. Scattered Schwann cells were alsoseen in regions without growing axons. Schwann cell migration wasapparent well into the 8-day grafts. However, at more distal points inthe grafts, axons were often found without accompanying Schwann cells,as shown in FIG. 7. This was confirmed in longitudinal sectionsincluding the distal coaptation, as shown in FIG. 8. S-100 labeledSchwann cells were abundant in the distal host stumps, yet few if anyhad invaded the distal aspect of the grafts (which contained onlyfreeze-killed Schwann cell remnants), as shown in FIG. 8B. The examplespresented in FIGS. 7, 8A, and 8B, were obtained fromchondroitinase-treated grafts and identical results were observed in thecontrol grafts. These findings suggested that the enhancement of axonalgrowth in chondroitinase-treated grafts was primarily attributed to thepotentiation of the neurite-promoting activity of the basal lamina.

The path of axonal growth was examined only in longitudinal sections oftissues immediately surrounding the proximal and distal coaptations.Upon entering the grafts, axon growth was directed distally and therewas no indication of deviant growth or neuroma formation within thegrafts. This suggested that guidance mechanisms (or chemoattractantproperties associated with the distal stump) were not compromised inchondroitinase-treated grafts. In addition, based on the few instanceswhere axons had reached the distal extent of the graft, axons exited thegrafts and continued growth into the host nerve stump, as shown in FIG.8A.

Example 6—Degradation and Inactivation of Inhibitory CSPG by Treatmentof Acellular Human Nerve Segments with Chondroitinase

Many of the experiments described in the examples above performed usingrat nerves also have been replicated using human nerve. Except whereotherwise indicated, the procedures described in Examples 1 and 2 usingrat nerve were also followed in Examples 6 and 7 using human nerve.Human sural and tibial nerves were obtained fresh from surgical legamputation. Amputations were necessary for diseases that did not havenerve involvement (e.g., bone cancer) and nerves were judged to benormal on the basis of histological examination.

Human nerves were first examined to determine if their content of CSPGand laminin was similar to that observed in rat nerves.Immunocytochemistry showed that the basal lamina, which supports nerveregeneration, contained both CSPG and laminin, which are colocalized inthe same fashion as in other species. Human nerves stained for CSPGneoepitope and laminin are shown, respectively, in FIGS. 9A and 9B.Furthermore, using the cryoculture bioassay, it was found that thegrowth-promoting properties of human nerves were increased by treatmentwith chondroitinase. Quantitative results are shown in FIG. 10.

Example 7—Axon Regeneration within Chondroitinase-Treated Human NerveGrafts

In the following experiments, the hypothesis that chondroitinasetreatment improves nerve regeneration through acellular human nervegrafts in a rat xenograft model was tested. Subunits (individualfascicles) taken from human nerves were treated en bloc with vehicle orchondroitinase ABC. Ten-mm interpositional nerve grafts were joined tothe rat host sciatic nerve by epineurial neurorrhaphy reinforced withfibrin glue. Each of 2 host rats received bilateral grafts, onevehicle-treated and one chondroitinase-treated graft. Regeneration wasinitially examined after 8 days. Axonal growth was assessed at specifiedspatial intervals within the graft by scoring GAP-43-immunopositiveprofiles in transverse sections.

The growth into chondroitinase-treated grafts was markedly greater andmore widely distributed than in control grafts. Quantitative results areshown in FIG. 11. The average number of axons (GAP-43-immunopositiveprofiles) entering chondroitinase-treated grafts was on average morethan three-fold greater than in control grafts. These findings indicatethat the success of axonal penetration into acellular human nerve graftswas markedly improved by pretreatment of the grafts with aCSPG-degrading enzyme. This xenograft model also demonstrates thatacellular human nerves were not rejected (within 8 d) by the rat host,confirming the low immunogenicity of acellular nerves.

Example 8—Degradation of CSPG in the Nerve after ChondroitinaseInjection

Animals received either bilateral nerve crush injury or bilateral nervetransection and direct suture repair. At the same time, one nerve wasinjected with chondroitinase ABC and the contralateral nerve receivedvehicle alone. Whether the chondroitinase treatment effectively degradedCSPG in the injured nerves was first examined. Tissue sections of nerveat and surrounding the site of injury were immunolabeled usingCSPG-neoepitope antibodies. These antibodies bind to new epitopescreated on the CSPG core protein after lysis of chondroitin sulfatechains by chondroitinase ABC. In transected nerves four days afterinjury and chondroitinase application, CSPG-neoepitope immunostainingwas intense at the coaptation and throughout the cross-sectional area ofthe distal nerve several mm peripherally, as shown in FIGS. 12A and 12B.Intense immunolabeling was also observed several mm into the proximalnerves. Similar results were obtained in crush-injured nerves, as shownin FIG. 12C. CSPG-neoepitope labeling of tissue near the coaptation andinjection site was at most marginally more intense when a secondarytreatment with chondroitinase was applied to the tissue sections as partof the immunostaining procedure, as shown in FIG. 12D. These resultsindicate that in vivo treatment with chondroitinase ABC substantially,if not completely, degraded CSPG in the extracellular matrix surroundingthe site of injection, including the site of nerve injury and repair.

Example 9—Chondroitinase Treatment Did not Alter Axonal Regenerationafter Nerve Crush Injury (Axonotmesis)

The hypothesis that chondroitinase treatment improves axonal growththrough the site of nerve crush injury was tested. Bilateral axonotmesiswas achieved by severely crushing the sciatic nerves while maintainingthe continuity of the nerve sheaths. At the time of injury, one nervewas injected with chondroitinase ABC and the contralateral nervereceived vehicle alone. Because of the rapid regrowth of axons afternerve crush, axonal growth across the injury site was examined two daysafter injury. Regenerating axons were labeled by GAP-43immunofluorescence and scored by digital image analysis. As expected incontrol nerves, axonal regeneration directly distal to the crush sitewas robust and widespread throughout the nerve cross-section, as shownin FIG. 13A. A similar regenerative response and growth pattern wasobserved in the chondroitinase-treated nerves as well. In bothconditions, immunolabeling was very dense and numerous neurites wereseen within each basal lamina tube. Quantitative assessment of GAP-43immunoreactivity showed that axonal regeneration after nerve crushinjury was not significantly effected by chondroitinase application(FIG. 13B). Likewise, there was no indication that the latency or rateof axonal regeneration was altered in the chondroitinase-treated nerves.

Example 10—Regeneration of Axons after Nerve Transection (Neurotmesis)Repair is Enhanced by Chondroitinase Treatment

The hypothesis that chondroitinase treatment improves axonal growththrough the site of nerve coaptation was tested. Bilateral neurotmesiswas achieved by a scissor cut of the sciatic nerves which were thenrepaired by epineurial suture and fibrin glue. One nerve was injectedwith chondroitinase ABC and the contralateral nerve received vehiclealone. Because of the latency of regeneration after nerve transection,axonal growth across the coaptation was examined four days after injury.In control nerves, the ingress of axons occurred mainly in patches andwas limited to discrete subsections of the distal nerve; otherwise onlya few axons were found scattered throughout the remaining nervecross-section, as shown in FIG. 14A. The number of axons extendingfarther into the distal (after 4 days) rapidly diminished and approachedzero within 3 mm from the coaptation. In contrast, axon ingress intochondroitinase-treated nerves was more robust and widespread throughoutthe entire nerve cross-section. In 7/7 animals, the number of axons thatentered the distal nerve was greater in the chondroitinase-treated nervethan in the control nerve. On average, the score of axons immediatelydistal to the coaptation was two-fold greater in thechondroitinase-treated nerves, and 3/7 animals had increases greaterthan 3.5-fold, as shown in FIG. 14B. The ratio of axon numbers inchondroitinase-treated compared to control nerves progressivelyincreased from 2:1 (just beyond the coaptation) to more than 4:1 atpoints farther into the distal stump. Thus, in addition to increasingthe number of axons invading the distal nerve, chondroitinase treatmentalso decreased the latency of axonal ingress and/or increased the rateof growth within the distal nerve segment. It was clear that axon growthin all portions of the distal nerve was strictly linear and aligned withthe longitudinal axis of the nerve. In addition, double-immunolabelingfor regenerating axons (GAP-43) and basal laminae (laminin) indicatedthat the strong propensity of axons to regrow within basal laminae ofthe distal nerve was unaltered by chondroitinase treatment.

These findings show that axonal regrowth after crush injury was similarin chondroitinase-treated and control nerves. In contrast, axonalregrowth through the coaptation of transected nerves was accelerated andthe ingress of axons into the distal segment was increased several-foldin nerves injected with chondroitinase. Thus, in transection injury whennerve continuity is disrupted, chondroitinase application significantlyincreased the ability of axons to access basal laminae of the distalnerve segment and markedly enhanced regeneration.

In accordance with the subject invention, a single injection ofchondroitinase can markedly improve axonal regeneration across theinterface of coapted peripheral nerve. Degradation of inhibitory CSPGcreates a more permissive nerve substratum and allows axon sproutsgreater latitude in their effort to locate and access Schwann cell basallaminae of the distal nerve. The difficulty that axons face in thisprocess is evidenced by the increased latency associated withregeneration after transection injury as compared to crush injury.Notably, the data suggest that an important effect of chondroitinasetreatment is to decrease the latency of regeneration in the peripheralnerve transection model. In addition, it is known that axonal sproutswill degenerate if they fail to traverse the coaptation (Fu, S. Y., andT. Gordon 1997, Mol Neurobiol 14: 67-116).

Example 11—The Neurite-Promoting Activity of Cultured Nerve Segments

Freshly excised (cellular) rat sciatic nerve segments were cultured forup to 7 days in medium containing 0, 2 and 10% fetal bovine serum.Control (uncultured) and cultured nerves were cryosectioned and theirneurite-promoting activity was assessed by cryoculture assay. Resultsare shown in FIG. 15A. Embryonic chick DRG neurons grown on sections ofcontrol nerves had an average neurite length of 118 μm. Neuritic growthon sections of nerve explants cultured for 1-4 days was significantlygreater. For nerves cultured in defined medium (0% serum),neurite-promoting activity reached a maximum at 2 days in vitro,representing a 43% increase compared to control nerves. There was morethan a 70% increase in the neurite-promoting activity for nerve explantscultured for 1 or 2 days in medium containing 2% serum. Nerve explantscultured in 10% serum reached a similar maximum at 2 days in vitro aswell. The neurite-promoting activity of nerves explants declined afterlonger culture periods and fell below the level of the control conditionat 7 days. These data indicate that the neurite-promoting activity ofnerve explants increased markedly when cultured for short periods invitro with and without the addition of serum to the culture medium.Nerve explants were prevented from adhering to the culture vessel and nocell outgrowth was observed. However, cell viability in all conditionswas confirmed in separate experiments in which robust cell migration wasobserved from nerve explants that were minced and pressed to the culturesurface.

Example 12—Comparison of In Vitro and In Vivo Predegeneration

Using the cryoculture assay, the neurite-promoting activity of ratsciatic nerves predegenerated in vitro was compared to thosepredegenerated in vivo. As described above (see FIGS. 15A and 15B),neuritic growth of DRG neurons on nerve explants cultured for 2 days in2% serum (in vitro predegeneration) was 70% greater than control nerves(not predegenerated). Also, nerve explant culture for longer periods (4and 7 days) resulted in a progressively less neurite-promoting activity.Nerves cultured for 7 days had 37% less activity than the controlcondition. By comparison, the neurite-promoting activity of nervespredegenerated in vivo was much lower those predegenerated in vitro.Neuritic growth on nerves predegenerated in vivo for 2 days was 35.8 μm,72% less activity than the control condition (126.5 μm) (t-test,p<0.001). However, this inhibition was reversed over time and in vivopredegeneration for 7 days resulted in neuritic growth 12% greater thanthe control condition (p=0.06). These data show that in vitropredegeneration increased the neurite-promoting activity of nervesegments to a greater extent than that achieved by in vivopredegeneration.

Example 13—In Vitro Degeneration is MMP-Dependent

Nerve segments were cultured for 2 days in medium containing 2% serumwith and without the addition of the MMP inhibitor, GM6001. Theneurite-promoting activity of the cultured nerves was assessed bycryoculture assay. Results are shown in FIG. 15B. Similar to that shownin FIG. 15A, the mean neurite length of DRG neurons grown on culturednerves (2-day, 2% serum) was 210 μm, representing a 68% increase overthat of (uncultured) control nerves. However, this increase was reducedto only 14% for nerves cultured in the presence of GM6001. Dissociationculture (squash preparations) of the nerve segments in each conditionshowed profuse cell outgrowth indicating no loss of cell viability. Inaddition, treatment of isolated Schwann cell cultures with GM6001confirmed the very low toxicity of this hydromate-based dipeptide. Theseresults strongly implicate MMP activity in a degenerative process thatincreases the neurite-promoting activity of cultured nerve explants.

Example 14—MMP Expression in the Cultured Nerve Segments: ZymographicGel Analysis

MMP-2 and MMP-9 are the main extracellular proteinases capable ofdegrading gelatin (cleaved collagen) and their major substrate iscollagen type IV of the basal lamina. MMP-2 is constitutively expressedby Schwann cells in vivo and is upregulated after nerve injury in therat. On the other hand, MMP-9 is undetectable in normal nerve and ispresent after injury in association with invading granulocytes andmacrophages. Examination of in vitro nerve degeneration in the presentinvention provides a unique opportunity to determine the role of MMPexpression by resident nerve cells with a minimal contribution byhematogenic cells. MMP levels in cultured nerve explants were firstexamined by gelatin substrate-overlay gel electrophoresis (zymography).Gelatin zymography is very sensitive in the detection of MMP-2 and MMP-9and has the added advantage of revealing both latent and activatedforms. Nerve segments were cultured for 1, 2, 4 and 7 days in thepresence of 2% serum A representative zymographic analysis of extractednerves is shown in FIG. 16. Normal (uncultured control) nerve showed apredominant gelatinolytie band at M_(r)=72 kD that comigrated with theproform of human recombinant MMP-2. A trace of activated MMP-2 wasobserved (M_(r)=66 kD), whereas MMP-9 (M_(r)=92 and 84 kD) was notdetected. In the cultured nerves, there was a rapid increase inactivated MMP-2 and a substantial increase in total MMP-2 content. MMP-9was undetectable in nerves cultured for 1 or 2 days and trace amounts ofactivated MMP-9 in the 4- and 7-day samples. Similar results wereobtained for nerve explants cultured in defined medium, confirming thatserum did not contribute to the gelatinolytic activity observed in thenerve samples. These findings indicate that MMP-2 is rapidly activatedand upregulated in nerve degeneration in vitro. It is notable thatgelatin zymography is several-fold more sensitive in detecting MMP-9than MMP-2 (Ladwig et al. [2002] Wound Repair Regen 10:26-37),signifying that MMP-9 content in the nerve samples was negligible.

Example 15—MMP Activity in the Cultured Nerve Segments: In SituZymography

The activity of MMPs is regulated by gene transcription, proenzymeactivation and by the action of tissue inhibitors of metalloproteinases.The net gelatinolytic activity in nerve segments by in situ zymographywas examined. Tissue sections were overlaid with quenched,fluorescein-gelatin, which is converted to fluorescent peptides bygelatinolytic activity within tissues. Constitutive gelatinolyticactivity was detected in normal nerve mostly associated with Schwanncells aligned along the endoneurial basal lamina (as shown in FIGS. 17Aand 17B). In cultured nerves there was widespread increase ingelatinolytic activity that was diffuse within the endoneurium andSchwann cells were labeled more intensively, as shown in FIGS. 17C and17D. Also examined, was the gelatinolytic activity in the nervescultured in the presence of GM6001. As described above, GM6001 blockedthe increases in neurite-promoting observed in cultured nerves.Gelatinolytic activity in GM6001-treated nerve explants was nearlyundetectable, as shown in FIGS. 17E and 17F. Together these findingsindicate that gelatinolytic activity was markedly increased by nerveexplant culture and that GM6001 effectively blocked de novo MMP activityduring in vitro degeneration.

Example 16—MMP Localization in the Cultured Nerve Segments:Immunofluorescent Labeling

The distributions of MMP-2 and MMP-9 in nerve explants cultured for 2days were examined by immunofluorescence microscopy. MMP-2immunolabeling of culture nerves was intense within Schwann cells andthe surrounding basal laminae, as shown in FIG. 18A. Schwann cellstaining with 5-100 indicated that the most intense MMP-2 immunolabelingwas associated with migrating Schwann cells (FIG. 18B; and see below).Also, MMP-2 immunoexpression was very similar to the pattern ofgelatinolytic activity obtained by in situ zymography. On the otherhand, MMP-9 immunolabeling was virtually absent within the nervefascicles, except for rare cellular profiles. Some cellularimmunoexpression of MMP-9 was seen in the surrounding epineurium, asshown in FIG. 18C. OX42 labeling was used to identify macrophages whichwere scattered throughout the epineurium and rarely within the nervefascicles of cultured nerves, as shown in FIG. 18D. The compartmentaldistributions of MMP-9 and OX42 labeling suggested that macrophages werethe main source of MMP-9. In addition, Schwann cells, and perhaps someperineurial fibroblasts, expressed MMP-2 and MMP-2 immunoreactivity wasalso observed diffusely in the surrounding extracellular matrix.

Example 17—Cell Distributions and Axonal Degeneration in the CulturedNerve Segments

After nerve injury Schwann cells become activated, dissociate theirmyelin and migrate extensively. S-100 immunolabeling of the culturednerve explants showed that many Schwann cells had lost their elongatedmorphology and close association with axons, typical of the injuryresponse, as shown in FIG. 18B. As expected when disconnected from thecirculatory system, the number of macrophages in the nerve explants wasmuch lower than that observed in nerve degeneration in vivo. Moreover,very few macrophages were found within the nerve fascicles and nearlyall OX42-labeled cells were confined to the epineurium, as shown in FIG.18D. It was clear that the macrophages present in the epineurialcompartment at the time of nerve excision did not invade the inner nervecompartments during culture. Accordingly, the nerve explants in vitrorepresent a model of nerve degeneration in which the contribution ofSchwann cells may be assessed independently from those of invadingmacrophages.

The degradation of axons was examined in cultured nerve explants byimmunolabeling of neurofilaments. Results are shown in FIGS. 19A-19D.Unlike the contiguous neurofilament staining observed in normal nerve,shown in FIG. 19A, the neurofilament profiles in nerve segments culturedfor 2 days were fragmented and irregular, as shown in FIG. 19B. Similarto axonal degeneration in vivo, the cultured nerves contained bothannular and condensed neurofilament profiles, indicative of cytoskeletondisintegration and axonal degeneration. The degeneration of axons wasespecially obvious in semi-thin sections stained with toluidine bluewhich showed a void or a dense pellet within the residual myelinsheaths, as shown in FIG. 19D. The degenerative changes observed in thenerves cultured for 2 days were reminiscent of the initial phase ofWallerian degeneration seen in vivo (reviewed by Stoll and Muller, 1999,Brain Pathol 9:313-325). The main features of the secondary phase ofWallerian degeneration were also observed in cultured nerves includingmorphologic changes in the myelin sheath and myelin extrusion by Schwanncells, as well as Schwann cell proliferation, as shown in FIG. 19D.However, the degenerative processes resulting in further myelindegeneration (collapse and condensation) and phagocytotic removal didnot occur in the 2-day nerve explant cultures. Despite the substantialdegenerative alterations, the basal lamina scaffold remainedstructurally intact and remodeling was indicated by the high level oflaminin expression by Schwann cells, as shown in FIG. 19C.

Example 18—Cultured Nerve as Acellular Interpositional Grafts

The present experiment tests the hypothesis that predegeneration invitro improves nerve regeneration through acellular nerve allografts.Host rats received bilateral, acellular nerve grafts, one control (notpredegenerated) and one predegenerated in vitro (cultured for 2-day in2% serum). Axonal regeneration was assessed after 8 days by scoringGAP-43-immunopositive profiles in transverse sections. Axonal growth wasobserved in all grafts and was centrally distributed, indicating goodalignment and coaptation of proximal host nerve and graft, as shown inFIGS. 20A and 20B. In 6/6 animals, the number of axons that crossed theproximal nerve-graft coaptation and entered the graft was greater in thein vitro predegenerated graft than in the contralateral control graft.On average, the score of axons within the in vitro predegenerated graftswas two-fold greater, as shown in FIG. 20B. In both graft conditions,axonal growth occurred within basal lamina tubes and was accompanied byhost derived Schwann cells. These findings show that axonal regenerationinto acellular nerve grafts is enhanced by in vitro predegeneration.

Degeneration increases the growth-promoting properties of denervatedperipheral nerve and derivative nerve grafts. The present experimentinvestigated the role of MMPs in this degenerative process using a nerveexplant culture model. Also, because nerve predegeneration in vivo isnot feasible for the preparation of human allografts, the attributes ofnerve grafts predegenerated in vitro were examined. The results of thepresent experiment support the following conclusions. First, earlystages of Wallerian degeneration occur in short-term culture ofperipheral nerve explants, despite the absence of hematogenicmacrophages. The neurite-promoting activity of nerve segments ismarkedly increased by in vitro degeneration and to a greater extent thannerve predegenerated in vivo. The increase in neurite-promoting activityresulting from in vitro degeneration is attributed to a heightenedexpression and activation of MMP-2 by Schwann cells. Lastly, in vitropredegeneration enhances axonal regeneration into acellularinterpositional nerve grafts.

The present experiment of peripheral nerve degeneration in vitro, findsthat MMP-9 is present in trace amount mostly associated with a minorpopulation of cells restricted to the epineurial sheath. Immunolabelingfor MMP-9 is essentially absent in the endoneurial compartment ofcultured nerves. In contrast, MMP-2, particularly the activated form,rapidly increases within the endoneurium in cultured nerves. Takentogether with immunolocalization and in situ zymography data, theexperimental data concludes that MMP-2 is expressed by Schwann cells andactive enzyme is released into the surrounding endoneurium during invitro nerve degeneration.

Combined with the present observations of nerve explants, theexperimental data shows that MMP-2 represents a sufficient, if notprincipal, degenerative mechanism for the enhancement of thegrowth-promoting properties of denervated nerve (and predegeneratednerve grafts).

According to the current invention, culture of nerve explants, usingconditions to support cell viability and growth, allows forcell-mediated degeneration and significantly enhances the regenerativepotential of nerve grafts. Nerve explants can be freeze-killed andstored frozen for later use as interpositional nerve grafts.Freeze-killing nerve grafts virtually eliminates the concerns of graftimmunorejection. For this reason acellular nerve grafts have a greaterpotential for clinical applications than do cellular nerve grafts inallografting without immunosuppression.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

The invention claimed is:
 1. A human peripheral nerve graft, suitablefor implantation into a human to coapt a severed peripheral nerve,wherein said graft is prepared by a method comprising treating humanperipheral nerve graft tissue with a detergent to render the human nervegraft tissue acellular and further applying at least one chondroitinaseto said human nerve graft tissue, wherein the resulting nerve graftcomprises an intact basal lamina tube and is capable of supportingaxonal ingress after implantation into a human to coapt a severed nerve,wherein the treatment with the chondroitinase conditions the nerve graftfor enhanced post-implantation axonal ingress such the post-implantationaxonal ingress is enhanced relative to a nerve graft to which thechondroitinase has not been applied.